Assays for the detection of microtubule depolymerization inhibitors

ABSTRACT

This invention provides assays for agents that modulate (e.g. upregulate, downregulate or completely inhibit) microtubule depolymerizing or microtubule severing proteins. Such agents will have profound effects on progression of the cell cycle and act as potent anti-mitotic agents. The microtubule severing protein or microtubule depolymerizing protein is preferably a katanin, a p60 subunit of a katanin, an XKCM 1, or an OP18 polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of copending Application Ser. No. 09/673,222,filed Oct. 13, 2000, which is the U.S. National stage entry filing, andwhich claims the benefit under 35 U.S.C. §119(e), of PCT/US99//08086,filed Apr. 13, 1999 which claims priority to U.S. Provisional PatentApplication Serial No. 60/081,734, filed Apr. 14, 1998, now abandoned,which is herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[Not Applicable ]

FIELD OF THE INVENTION

This invention relates assay for agents that modulate (e.g. upregulate,downregulate or completely inhibit) microtubule depolymerizing ormicrotubule severing proteins. Such agents will have profound effects onprogression of the cell cycle and act as potent anti-mitotic agents.

BACKGROUND OF THE INVENTION

The cytoskeleton constitutes a large family of proteins that areinvolved in many critical processes of biology, such as chromosome andcell division, cell motility and intracellular transport. Vale andKreis, 1993, Guidebook to the Cytoskeletal and Motor Proteins New York:Oxford University Press; Alberts et al., (1994) Molecular Biology of theCell, 788-858). Cytoskeletal proteins are found in all cells and areinvolved in the pathogenesis of a large range of clinical diseases. Thecytoskeleton includes a collection of polymer proteins, microtubules,actin, intermediate filaments, and septins, as well as a wide variety ofproteins that bind to these polymers (polymer-interacting proteins) Someof the polymer-interacting proteins are molecular motors (myosins,kinesins, dyneins) (Goldstein (1993) Ann. Rev. Genetics 27: 319-351;Mooseker and Cheney (1995) Annu. Rev. Cell Biol. 11: 633-675) that areessential for transporting material within cells (e.g., chromosomalmovement during metaphase), for muscle contraction, and for cellmigration. Other groups of proteins (e.g., vinculin, talin andalpha-actinin) link different filaments, connect the cytoskeleton to theplasma membrane, control the assembly and disassembly of thecytoskeletal polymers, and moderate the organization of the polymerswithin cells.

Given the central role of the cytoskeleton in cell division, cellmigration, inflammation, and fungal/parasitic life cycles, it is afertile system for drug discovery. Although much is known about themolecular and structural properties of cytoskeletal components,relatively little is known about how to efficiently manipulatecytoskeletal structure and function. Such manipulation requires thediscovery and development of specific compounds that can predictably andsafely alter cytoskeletal structure and function. However, at present,drug targets in the cytoskeleton have been relatively untapped.Extensive work has been directed towards drugs that interact with thecytoskeletal polymers themselves (e.g., taxol and vincristine), andtowards motility assays. Turner et al. (1996) Anal. Biochem. 242 (1):20-5; Gittes et al. (1996) Biophys. J. 70 (1): 418-29; Shirakawa (1995)J. Exp. Biol. 198: 1809-15; Winkelmann et al. (1995) Biophys. J. 68:2444-53; Winkelmann et al. (1995) Biophys. J. 68: 72S. Virtually noeffort has been directed to finding drugs that target the cytoskeletalproteins that bind to the different filaments, which might be morespecific targets with fewer unwanted side effects.

SUMMARY OF THE INVENTION

This invention pertains to the discovery that proteins (e.g. motorproteins) that either depolymerize or sever microtubules, provide goodtargets for modulators of such activity. Without being bound by aparticular theory, it is believed that microtubule depolymerizing orsevering activity is critical for normal formation and/or function ofthe mitotic spindle. Thus, agents that modulate (e.g., upregulate,downregulate, or completely inhibit) depolymerization or severingactivity are expected to have a significant activity on progression ofthe cell cycle (e.g. acting as potent anti-mitotic agents).

*This invention thus provides, in one embodiment, assays for identifyingan agent that modulates microtubule depolymerization. The assays involvecontacting a polymerized microtubule with a microtubule severing proteinor a microtubule depolymerizing protein in the presence of an ATP or aGTP and the “test” agent; and detecting the formation of tubulinmonomers, dimers or oligomers. The microtubule can be labeled with anyof a variety of labels, however in a preferred embodiment, it is labeledwith DAPI. The formation of tubulin monomers, dimers, or oligomers canbe detected by any of a wide variety of methods including, but notlimited to changes in DAPI fluorescence, fluorescent resonance energytransfer (FRET), centrifugation, and the like. The microtubules arepreferably microtubules that are either naturally stable (e.g. axonemalmicrotubules) or microtubules that have been stabilized e.g. by contactwith an agent such as paclitaxel, a paclitaxel analogue, or anon-hydrolyzable nucleotide GTP analogue.(e.g., guanylyl-(α,β)-methylenediphosphate (GMPCPP)).

The assays can be run in solution or in solid phase (i.e. where one ormore assay components are attached to a solid surface. In oneembodiment, of solid-phase assays, the microtubule is attached to thesurface e.g. by direct binding or by binding with an agent such as aninactivated microtubule motor protein, an avidin-biotin linkage, ananti-tubulin antibody, a microtubule binding protein (MAP), or apolylysine. The microtubule severing protein or microtubuledepolymerizing protein is preferably a katanin, a p60 subunit of akatanin, an XKCM1, or an OP18 polypeptide. In a particularly preferredembodiment, the microtubule severing protein is a katanin or a p60subunit of a katanin as described herein.

It was also a discovery of this invention that the katanin p60 subunitexhibits both the ATPase and microtubule severing activity observed inkatanin. The p60 subunit thus provides a good target for screening forpotential therapeutic lead compounds Thus, in another embodiment, thisinvention provides methods screening for (identifying) a therapeuticlead compound that modulates depolymerization or severing of amicrotubule system. The methods involve providing an assay mixturecomprising a katanin p60 subunit and a microtubule, contacting the assaymixture with a test compound to be screened for the ability to inhibitor enhance the microtubule-severing or ATPase activity of the p60subunit; and detecting specific binding of the test compound to said p60subunit or a change in the ATPase activity of the p60 subunit. Thedetecting can be by any of a wide variety of methods including, but notlimited to detecting ATPase activity using malachite green as adetection reagent. Binding activity can be easily detected in bindingassays in which the p60 subunit is labeled and said test agent isattached to a solid support or conversely, the test agent is labeled andthe p60 subunit is attached to a solid support. In a preferredembodiment, the ATPase assays are performed in the presence ofstabilized microtubules.

The assay methods of this invention are also amendable to highthroughput screening. Thus, in one embodiment, any of the methodsdescribed herein is performed in an array where said array comprises amultiplicity of reaction mixtures, each reaction mixture comprising adistinct and distinguishable domain of said array, and wherein the assaysteps are performed in each reaction mixture. The array can take anumber of formats, however, in one preferred format, the array comprisesa microtitre plate, preferably a microtitre plate comprising at least 48and more preferably at least 96 reaction mixtures. The test agent can beone of a plurality of agents and each reaction mixture can comprises oneagent of the plurality of agents.

In addition, this invention provides for polypeptides having microtubulesevering activity. The polypeptides comprise an isolated p60 subunit ofa katanin, where the p60 subunit is encoded by a nucleic acid thathybridizes under stringent conditions with a nucleic acid that encodesthe katanin p60 amino acid sequence (SEQ ID NO: 1). In a particularlypreferred embodiment, the polypeptide is the polypeptide of SEQ ID NO: 1or the polypeptide of SEQ ID NO: 1 having conservative substitutions.The polypeptide can comprise at least 8 contiguous amino acids from apolypeptide sequence encoded by a nucleic acid as set forth in SEQ IDNO: 1, where the polypeptide, when presented as an antigen, elicits theproduction of an antibody that specifically binds to a polypeptidesequence encoded by a nucleic acid as set forth in SEQ ID NO: 1; and thepolypeptide does not bind to antisera raised against a polypeptideencoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, thathas been fully immunosorbed with a polypeptide encoded by a nucleic acidsequence as set forth in SEQ ID NO: 1. In a most preferred embodiment,the polypeptide is polypeptide of SEQ ID No: 1.

This invention also provides an isolated nucleic acid that encodes akatanin p60 subunit having microtubule severing activity. The nucleicacid preferably comprises a nucleic acid that specifically hybridizeswith a nucleic acid that encodes the polypeptide of SEQ ID NO:1 understringent conditions. The nucleic acid preferably encodes a polypeptideof SEQ ID No: 1 or conservative substitutions thereof. The katanin p60encoding nucleic acid can be operably linked to a promoter (e.g. abaculovirus promoter) and may be present in a vector.

In another embodiment, this invention provides methods of screening foran agent that alters microtubule polymerization, or depolymerization, orsevering. The methods involve providing labeled tubulin; contacting thelabeled tubulin with the test agent to produce contacted tubulin; andcomparing the fluorescence intensity or pattern of the contacted tubulinwith the fluorescence intensity or pattern of labeled tubulin that isnot contacted with the test agent where a difference in fluorescencepattern or intensity between the contacted and the not contacted tubulinindicates that the agent alters microtubule polymerization, ordepolymerization, or severing. In particularly preferred embodiments,the labeled tubulin is in the form of tubulin monomers, tubulin dimers,tubulin oligomers, or a microtubule. In some embodiments, themicrotubule is attached to a solid surface (e.g., by by binding with anagent selected from the group consisting of an inactivated microtubulemotor protein, an avidin-biotin linkage, an anti-tubulin antibody, amicrotubule binding protein (MAP), a polyarginine, a polyhistidine, anda polylysine). Preferred labels include DAPI, ANS, Bis-ANS, rutheniumred, cresol violet, and DCVJ, with DAPI being most preferred. In someembodiments, the “contacting” step can further comprise contacting thetubulin with a microtubule depolymerizing protein or a microtubulesevering protein. Preferred microtubule severing or a microtubuledepolymerizing proteins include, but are not limited to katanin, a p60subunit of a katanin, an XKCM1, and a OP18 polypeptide. A preferred p60subunit of a katanin is a polypeptide of SEQ ID NO: 1. The method canfurther involve listing the agents that alters microtubulepolymerization, depolymerization, or severing into a database oftherapeutic lead compounds that act on the cytoskeletal system. Thismethod can be performed in various array embodiments as describedherein.

This invention also provides kits for practice of any of the methodsdescribed herein. In one embodiment, the kits comprise one or morecontainers containing an isolated microtubule severing protein or amicrotubule depolymerizing protein. The kit can further comprise apolymerized microtubule labeled with DAPI. The microtubule can bestabilized by contact with paclitaxel or a paclitaxel derivative. Themicrotubule can also optionally be attached to a solid surface (e.g., bybinding with an inactivated motor protein). The microtubule severingprotein or microtubule depolymerizing protein is preferably selectedfrom the group consisting of a katanin, a p60 subunit of a katanin, anXKCM1, and a OP18 polypeptide. In a particularly preferred embodiment,the microtubule severing protein is a katanin or a p60 subunit of akatanin.

DEFINITIONS

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides, and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated.

Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka etal. (1985) J. Biol. Chem. 260: 2605-2608; and Cassol el al. (1992);Rossolini et al., (1994) Mol. Cell. Probes 8: 91-98). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are preferably in the natural “L” isomeric form. However,residues in the “D” isomeric form can be substituted for any L-aminoacid residue, as long as the desired functional property is retained bythe polypeptide. In addition, the amino acids, in addition to the 20“standard” amino acids, include modified and unusual amino acids, whichinclude, but are not limited to those listed in 37 CFR ∃1.822(b)(4).Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates either a peptide bond to afurther sequence of one or more amino acid residues or a covalent bondto a carboxyl or hydroxyl end group.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The terms “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state. However, the term “isolated”is not intended refer to the components present in an electrophoreticgel or other separation medium. An isolated component is free from suchseparation media and in a form ready for use in another application oralready in use in the new application/milieu.

The terms “identical” or percent “identity,” or percent “homology” inthe context of two or more nucleic acids or polypeptide sequences, referto two or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, as measuredusing one of the following sequence comparison algorithms or by visualinspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95% or evenat least 98% amino acid residue identity across a window of at least 30nucleotides, preferably across a window of at least 40 nucleotides, morepreferably across a window of at least 80 nucleotides, and mostpreferably across a window of at least 100 nucleotides, 150 nucleotides,200 nucleotides or greater, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins & Sharp (1989) CABIOS 5:151-153. The program can align up to 300sequences, each of a maximum length of 5,000 nucleotides or amino acids.The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al. (1990) J. Mol. Biol. 215:403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extension of the word hits in each direction are halted when:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST algorithm parametersW, T, and X determine the sensitivity and speed of the alignment. TheBLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci.USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4,and a comparison of both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

The phrases “hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refer to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. AStringent hybridization≡and Astringent hybridization wash conditions≡ in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part 1 chapter 2 Aoverview of principles of hybridization and thestrategy of nucleic acid probe assays≡, Elsevier, N.Y. Generally, highlystringent hybridization and wash conditions are selected to be about 5EClower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Very stringent conditions areselected to be equal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42EC, with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72EC for about 15 minutes. An example of stringent wash conditions isa 0.2×SSC wash at 65EC for 15 minutes (see, Sambrook et al. (1989)Molecular Cloning-A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.) suprafor a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45EC for 15 minutes. An example low stringencywash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at40EC for 15 minutes. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

The terms “katanin” or “katanin p60 subunit” refer to katanin and thekatanin p60 subunit as described herein, in the references cited and inthe sequence listings. The terms also include proteins havingsubstantial amino acid sequence identity with katanin or the katanin p60subunit sequences provided herein that exhibit ATPase and microtubulesevering activity.

The terms “taxol” and “taxol derivatives or analogues refer to the drugtaxol known generically as paclitaxel (NSC number: 125973). Paclitaxel(taxol) derivatives and analogues show similar microtubule-stabilizingactivity. Preferred derivatives include taxotere and others.

Depolymerized microtubule components defined to include the products ofmicrotubule depolymerization or severing. Include tubulin monomers,dimers or oligomers.

The term “test agent” refers to an agent that is to be screened in oneor more of the assays described herein. The agent can be virtually anychemical compound. It can exist as a single isolated compound or can bea member of a chemical (e.g. combinatorial) library. In a particularlypreferred embodiment, the test agent will be a small organic molecule.

The term small organic molecules refers to molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes biological macromolecules (e.g., proteins, nucleicacids, etc.). Preferred small organic molecules range in size up toabout 5000 Da, more preferably up to 2000 Da, and most preferably up toabout 1000 Da.

The terms “label” or “detectable label” are used herein to refer to anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Such labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein,texas red, rhodamine, green fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and calorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Means of detecting such labels are well known to those ofskill in the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted light. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and calorimetric labels are detected by simplyvisualizing the colored label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A (SEQ ID NO: 1) and 1B (SEQ ID NOS: 4-9) show the sequenceanalysis of p60 katanin. FIG. 1A: Predicted protein sequence of the S.purpuratus katanin 60 subunit (GENBANK AF052191). Sequences obtained bydirect peptide microsequencing are underlined. Differences between thepredicted peptide sequence and that obtained by direct sequencing areindicated by double underlines (S95 was reported as F, H99 was reportedas P, and P138 was reported as T). The Walker A (P-loop) motif isshaded. FIG. 1B: Amino acid sequence alignment of the p60 AAA domainwith AAA members mei-1 (C. elegans, GenBank L25423), Suglp (S.cerevisiae, GenBank X66400), ftsH (E. coli, GenBank M83138), Paslp (S.cerevisiae), GenBank M58676), and NSF (C. longicaudatus, GenBankX15652). Identical residues are shaded black, residues conserved in >60%of the shown members are shaded gray. Left hand numbering indicates theamino acid residue in the corresponding sequence. Alignment wasperformed using PILEUP (Genetics Computer Group) and the output wasshaded using MACBOXSHADE.

FIGS. 2A (SEQ ID NO: 2) and 2B (SEQ ID NOS: 10-13) show the sequenceanalysis of p80 katanin. FIG. 2A: Predicted protein sequence of the S.purpuratus katanin p80 subunit (GENBANK AF052433). Sequences obtained bydirect peptide microsequencing are underlined. Differences between thepredicted peptide sequence and that obtained by direct peptidesequencing, or differences found between 2 different p80 cDNA clones areindicated by double underlines. FIG. 2B: Amino acid sequence alignmentof the WD40 repeat region of p80 with a putative human ortholog of p80(Hs p80, GenBank AF052432), TFIID (Homo sapiens, GenBank U80191), andputative serine/threonine kinase PkwA (Thermomonospora curvata, GenBankP49695). Identical residues are shaded black, residues found in at least2 sequences are shaded in grey. Left hand numbering indicates the aminoacid residue in the corresponding sequence. Alignment was performedusing PILEUP (Genetics Computer Group) and the output was shaded usingMACBOXSHADE.

FIG. 3 illustrates the results of expression and purification ofrecombinant katanin subunits. Panel A shows coomassie-stained SDS-PAGEanalysis of expressed katanin subunits. 6×His-tagged katanin subunitswere purified from lysates of baculovirus-infected insect cells bybinding to Ni²⁺-NTA Superflow followed by elution with imidazole, asdescribed in the Experimental Procedures. Cells were infected witheither p60 virus alone, p80 virus alone, or coinfected with equalamounts of p60 and p80 viruses. Panel B shows immunoprecipitationperformed on extracts of insect cells coinfected with p60- andp80-expressing baculoviruses using affinity-purified p60 antibodycrosslinked to protein A agarose. Proteins bound to the resin wereanalyzed by SDS-PAGE followed by staining with Coomassie. Thisimmunoprecipitate shows that baculovirus-expressed p60 and p80 form acomplex with equal stoichiometry.

FIG. 4 shows the structure of katanin as visualized by rotary-shadowingelectron microscopy. Panel A shows 14-16 nm diameter rings observed inpreparations of recombinant p60. Panel B shows single particles ofrecombinant p80; occasional aggregates are seen (rightmost picture) butrings are never observed. Panels C and 4D show different rings observedin recombinant p60/p80 preparations. .Panel C shows a “splayed” complex,consisting of a central p60-like ring surrounded by a halo particlesthat resemble p80. In Panel D, intact 20 nm diameter rings are seen withbright edges, suggesting they extend >10 nm above the mica surface. Allimages are shown at 300,000×. The dimensions indicated above include theplatinum shadowing, which typically adds 2 nm of material to the proteinsurface.

FIGS. 5A, 5B, and 5C illustrates the activities of recombinant kataninsubunits. FIG. 5A shows ATPase activities of 0.04 μM p60 katanin(squares) and co-expressed p60/p80 (circles) determined at variousmicrotubule concentrations as described in the Experimental Procedures.Both p60 katanin and p60/p80 show similar patterns of microtubulestimulation, with p60 katanin having approximately one half of themaximally-stimulated ATPase activity of p60/p80. The insert in the upperright shows the stimulation of ATPase activity at low (0-2 μM)microtubule concentration. FIG. 5B shows microtubule severing activityof recombinant katanin subunits. Taxol-stabilized, rhodamine-labeledmicrotubules were adsorbed onto the surface of a microscope perfusionchamber, and then recombinant katanin subunits were introduced. The timeelapsed after perfusing p60/p80 (0.1 μM), p60 (0.1 μM), or p80 (0.5 μM)is shown. The recombinant co-expressed p60/p80and p60, but not p80, cansever and disassemble microtubules. Scale bar,10 μm. FIG. 5C showsquantitative measurement of microtubule disassembly using a DAPIfluorescence assay. MT indicates microtubules (2 μM) without addedprotein, and tubulin indicates microtubules that had been depolymerizedby treatment with 10 mM CaCl₂ on ice for 1.5 hr. P60 katanin and p60/p80were added at 0.2 μM concentration, and the fluorescence change as afunction of time after protein addition is shown. p80 did not cause achange in fluorescence that was different from that shown formicrotubules alone.

FIG. 6 shows that the WD40 repeats of p80 katanin are not required forinteraction with p60 katanin. Epitope-tagged derivatives of p80 and p60were synthesized in vitro in a combined transcription-translationreaction. p60 and interacting proteins were immunoprecipitated with ap60-specific antibody and the resulting immunoprecipitates were resolvedby SDS-PAGE and blotted to nitrocellulose. In vitro translated proteinswere detected by chemiluminescence as described in ExperimentalProcedures. Lane. 1: molecular weight standards Mr: 100,000, 75,000,50,000, 35,000 and 25,000; lane 2: p60 co-translated with full-lengthp80; lane 3: p60 co-translated with the Δ560-690 derivative of p80; lane4: p60 co-translated with the Δ1-302 derivative of p80; lane 5: p60co-translated with the Δ303-690 derivative of p80. The structure of eachdeletion derivative of p80 is shown at right. The Δ560-690 and Δ303-690translation products were detected in the supernatants of theimmunoprecipitations (not shown).

FIG. 7 shows that human p80 katanin and a fusion protein of the humanp80 WD40 domain with GFP co-localize with γ-tubulin at centrosomes ofMSU1.1 human fibroblasts. Panel A and panel B: Co-localization ofimmunofluorescence staining by a human p80 katanin-specific antibody(FIG. 7A) and a γ-tubulin specific antibody (Panel B). Panels C-F showco-localization of GFP fluorescence (Panel C and Panel E) with stainingby a γ-tubulin-specific antibody (Panel D and Panel F). Co-localizationto two centrosomes is seen in Panel C and Panel D while co-localizationto a single centrosome is seen in Panel E and Panel F. The apparentlyhigher background of cytoplasmic green fluorescence in Panel E relativeto Panel C is a display artifact. The fluorescence intensity of thecentrosomes in Panel C is at least 5 fold greater than that of thecentrosome in Panel E. The p80 antibody was detected with an OregonGreen 488 second antibody and the γ-tubulin antibody was detected with aTexas Red-X second antibody. Fluorescence signals were separated withfluorescein and Texas Red filter sets (Chroma Technologies). Bar=14 μm.

DETAILED DESCRIPTION

I. Introduction.

This invention provides assays for the identification of agents thatmodulate the activity of microtubule depolymerizing or severingproteins. The assays generally involve contacting a polymerizedmicrotubule with a microtubule severing or depolymerizing protein (e.g.,XKCM1, OP18, katanin, etc.) the presence of a test agent and a chemicalenergy source (e.g., ATP or GTP). The effect of the agent on thedepolymerization or severing of the microtubules is then detectedtypically by detecting the formation of microtubule degradationcomponents (e.g. tubulin monomers, tubulin dimers, or tubulinoligomers). Test agents that alter the amount and or rate ofdepolymerization or severing of microtubules as compared to one or morecontrol assays are identified as modulators of microtubuledepolymerizing or severing activity.

It was a discovery of this invention certain proteins that eitherdepolymerize or sever microtubules, provide good targets for modulatorsof normal mitotic spindle formation. Without being bound by a particulartheory, it is believed that microtubule depolymerizing or severingactivity is critical for normal mitotic spindle formation and/orfunction. Agents that modulate (e.g., upregulate, downregulate, orcompletely inhibit) depolymerization or severing activity are expectedto have a significant activity on progression of the cell cycle. Thus,for example, inhibitors of microtubule depolymerization or severing willact as potent anti-mitotic agents.

Anti-mitotic agents are useful in a wide variety of contexts. Aspowerful anti-mitotics or anti-meiotics, the inhibitors of microtubuledepolymerizing or severing activity identified by the screening (assay)methods described herein, will have a wide variety of uses, particularlyin the treatment (e.g., amelioration) of pathological conditionscharacterized by abnormal cell proliferation. Such conditions include,but are not limited to: fungal infections, abnormal stimulation ofendothelial cells (e.g., atherosclerosis), solid tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas, vascular malfunctions(e.g., arterio-venous malformations), abnormal wound healing,inflammatory and immune disorders, Bechet's disease, gout or goutyarthritis, abnormal angiogenesis accompanying: rheumatoid arthritis,psoriasis, diabetic retinopathy, and other ocular angiogenic diseasessuch as retinopathy of prematurity (retrolental fibroplasic), maculardegeneration, corneal overgrowth, corneal graft rejection, neuroscularglaucoma, Oster Webber syndrome, and the like.

The inhibitors of microtubule depolymerization or severing will alsohave a variety of in vitro uses as well. For example, they can be usedto freeze cells in a particular stage of the cell cycle for a variety ofpurposes (e.g., in the preparation of samples for of histologicalexamination), in the isolation of nucleic acids from a particular stageof the cell cycle, and so forth.

The modulators identified by the assays of this invention are preferablycharacterized by specificity to the target microtubule depolymerizing orsevering proteins or the pathways characteristic of the activity ofthese proteins. They therefor provide novel lead compounds for thedevelopment of highly specific inhibitors for depolymerizing and/ormicrotubule severing protein families and subfamilies, thus allowing forprecise chemical intervention.

11. Assays for the Detection of Microtubule Depolymerization Modulators.

A) Depolymerization Assays

In one embodiment, this invention provides assays for thedetection/identification of agents that have activity in modulating thedepolymerization or severing of microtubules. The assays generallyinvolve contacting a polymerized microtubule with a microtubule severingprotein or a microtubule depolymerizing protein in the presence of achemical energy source (e.g., ATP or GTP) and said agent; and detectingand/or quantifying the formation of microtubule degredation products(e.g. tubulin monomers). Agents that inhibit the activity of themicrotubule depolymerizing or severing proteins will inhibit thebreakdown of the polymerized microtubules thereby delaying the formationof or reducing the quantity of tubulin monomers or oligomers. Thus adecrease in the rate of formation or amount of tubulin monomer or anincrease in the ratio of tubulin polymer (microtubule) to tubulinmonomer indicates an inhibitory modulating effect of the agent.Conversely, an increase in the rate of formation or amount of tubulinmonomer or an increase in the ratio of tubulin polymer (microtubule) totubulin monomer indicates a microtubule stabilizing modulating effect ofthe agent.

The increase or decrease is determined by reference to one or morecontrols. A control is essentially an identical assay that either lacksthe test agent or contains a “reference” agent that has a knownactivity. Assays lacking any test agent whatsoever act as negativecontrols, while assays utilizing an agent that has known modulatingactivity act as positive controls.

In a preferred embodiment, the assay is scored as positive (i.e., theagent has activity modulating a microtubule depolymerizing or severingprotein) when there is a significant difference between the negativecontrol and test assay and/or when there is no significant differencebetween the positive control and test assay. The significant differenceis preferably a statistically significant difference, more preferably atleast about a 10% difference, and most preferably at least about a 20%,30%, .50% or 100% difference.

The assays can be performed in solution or in solid phase (i.e. with oneor more components of the assay attached to a solid surface) asdescribed below. One particularly preferred embodiment is describedherein in Example I. The various components of the assay are describedbelow.

B) Binding Assays.

In another embodiment, this invention provides binding assays toidentify agents that inhibit binding of depolymerizing or severingproteins to microtubules or for agents that specifically bind to themicrotubule depolymerizing or severing polypeptide or polypeptidesubunit.

In preferred binding assays, the ability of the test agent tospecifically bind to the depolymerizing or severing protein is assayed.In a particularly preferred embodiment, the ability of the test agent tospecifically bind to a katanin P60 domain is assayed.

There are a wide variety of formats for binding assays. In oneembodiment, the depolymerizing or severing protein or protein subunit isimmobilized on a surface and contacted with the test agent or converselytest agent(s) are immobilized on a surface and specific binding of theprotein or protein subunit is assayed. Binding is most easily detectedwhere the moiety in solution (test agent or depolymerizing or severingprotein) is labeled and after the contacting and washing off of unboundagents, identification of the labeled moiety associated with the supportsuggests binding.

Solution phase binding assays are also known to those of skill in theart. For example, in one embodiment, the binding assay is acosedimentation assay. In this (pelleting) assay, when the test agentbinds to the microtubule severing or depolymerizing protein or proteinsubunits, the bound agent and protein will cosediment when centrifuged.Unbound polypeptide and test agent will either sediment at a differentrate or remain fully in solution.

Methods of performing various binding assays can be found in copendingapplication U.S. Ser. No. 60/057895 filed on Sep. 4, 1997. For a generaldescription of different formats for protein binding assays, includingcompetitive binding assays and direct binding assays, see Stites and A.Terr (1991) Basic and Clinical Immunology, 7th Edition; Maggio (1980)Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; and Tijssen (1985)Practice and Theory of Enzyme Immunoassays, in Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers, B. V.Amsterdam.

C) ATPase Assay.

It was a discovery of this invention that the katanin P60 subunit is anew member of the AAA family of ATPases and that expressed p60 hasmicrotubule-stimulated ATPase and microtubule-severing activities in theabsence of the p80 subunit. Thus, in another embodiment, this inventionprovides assays for agents that modulate the ATPase activity of akatanin p60 subunit.

ATPase assays are well known to those of skill in the art. In onepreferred embodiment, the assay can be performed according to themethods described by Kodama et al. (1986) J. Biochem. 99: 1465-1472.This assay, described in detain in Example 1, is performed with the testagent present and the results are compared to negative and/or positivecontrol assays to determine the ability of the test agent to alter(modulate) p60 ATPase activity.

D) Solid Phase Assays.

In one embodiment, the assays of this invention can be performed insolid phase where one or more components of the assay is attached to asolid surface. Solid phases assays, one or more components of the assayis attached to a solid surface. Virtually any solid surface is suitableas long as the surface material is compatible with the assay reagentsand it is possible to attach the component to the surface without undulyaltering the reactivity of the assay components. It is recognized thatsome components show reduced activity in solid phase, but this isgenerally acceptable so long as the activity is sufficient to detectand/or quantify depolymerization or severing activity of the subjectprotein.

Solid supports include, essentially any solid surface, such as a glassbead, planar glass, controlled pore glass, plastic, porous plasticmetal, or resin to which the molecule may be adhered. One of skill willappreciate that the solid supports may be derivatized with functionalgroups (e.g. hydroxyls, amines, carboxyls, esters, and sulfhydryls) toprovide reactive sites for the attachment of linkers or the directattachment of the component(s).

Adhesion of the assay component (e.g., microtubule(s)) to the solidsupport can be direct (i.e., the microtubule directly contacts the solidsupport) or indirect (a particular compound or compounds are bound tothe support, and the assay component binds to this compound or compoundsrather than to the solid support). The component can be immobilizedeither covalently (e.g., utilizing single reactive thiol groups ofcysteine for anchoring protein components, Colliuod et al. (1993)Bioconjugate Chem. 4, 528-536)), or non-covalently but specifically(e.g., via immobilized antibodies or other specific binding proteins(Schuhmann et al. (1991) Adv. Mater. 3: 388-391; Lu et al. (1995) Anal.Chem. 67: 83-87), the biotin/streptavidin system (Iwane et al. (1997)Biophys. Biochem. Res. Comm. 230: 76-80), or metal-chelatingLangmuir-Blodgett films (Ng et al. (1995) Langmuir 11: 4048-4055;Schmitt et al. (1996) Angew. Chem. Int. Ed. Engl. 35: 317-320; Frey etal. (1996) Proc. Natl Acad. Sci. USA 93:4937-494I; Kubalek et al. (1994)J. Struct. Biol. 113:117-123) and metal-chelating self-assembledmonolayers (Sigal et al. (1996) Analytical Chem., 68: 490-497) forbinding of polyhistidine fusion proteins.

In a preferred embodiment, the microtubule(s) are immobilized byattachment to an inactivated microtubule motor protein, by an avidinbiotin linkage (preferably with the biotin on the microtubule and theavidin on the surface), by an anti-tubulin antibody, by a microtubulebinding protein. (MAP), by an amino silane, a polylysine, or throughinteraction with a polycationic surface.

By manipulating the solid support and the mode of attachment of theassay component to the support, it is possible to control theorientation of the assay component(s). For example, copendingapplication U.S. Ser. No. 60/057,929, filed on Sep. 4, 1997, describesthe use of an arginine tail to attach cytoskeletal proteins to a micafilm.

In one preferred embodiment, the microtubules are immobilized by coatingthe surface (e.g., a flow cell) with either n-ethylmaleimide(NEM)-treated Xenopus egg extract (6 mg/ml protein treated with 10 mMNEM for 10 minutes followed by addition of 100 mM dithiothreitol, atreatment that inactivates severing activity) or Escherichiacoli-expressed KAR3 protein (which binds microtubules in anucleotide-independent manner. After washing out unbound protein, thestabilized microtubules (e.g. 100:g/ml in BR80, 20:M taxol) are perfusedonto the surface and allowed to bind. After washing out unboundmicrotubules samples to be tested can be contacted to the surface (e.g.,perfused into a flow cell).

E) High-throughout Screening of Candidate Agents that ModulateMicrotubule Depolymerizing or Severing Proteins.

Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of potential therapeuticcompounds (candidate compounds). Such “combinatorial chemical libraries”are then screened in one or more assays, as described herein, toidentify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

i) Combinatorial Chemical Libraries

Recently, attention has focused on the use of combinatorial chemicallibraries to assist in the generation of new chemical compound leads. Acombinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks called amino acids in every possible way for a given compoundlength (i.e., the number of amino acids in a polypeptide compound).Millions of chemical compounds can be synthesized through suchcombinatorial mixing of chemical building blocks. For example, onecommentator has observed that the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. (1994) 37(9): 1233-1250).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prof. Res., 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, Dec. 26,1991), encoded peptides (PCT Publication WO 93/20242, Oct. 14, 1993),random bio-oligomers (PCT Publication WO 92/00091, Jan. 9, 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta- D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries(see, e.g. U.S. Pat. No. 5,539,083) antibody libraries (see, e.g.,Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN,January 18, page 33, isoprenoids U.S. Pat. No. 5,569,588,thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974,pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholinocompounds U.S. Pat. No. 5,506,337, benzodiazepines U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,Hewlett-Packard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

ii) High Throughput Assays of Chemical Libraries

Any of the assays for compounds modulating the activity of microtubuledepolymerizing or severing proteins (or other agents) described hereinare amenable to high throughput screening. As described above, in apreferred embodiment, the assays screen for agents that enhance orinhibit the activity of katanin, XKCM1, or OMP19. Preferred assaysdetect the rate or amount of depolymerization of microtubules intotubulin monomers, tubulin dimers, or tubulin oligomers.

High throughput implementation of the assays described herein can beimplemented with, at most, routine modification of the assays format,e.g., for compatibility with robotic manipulators, large plate readers,and the like. Various high throughput screening systems (e.g., forprotein binding, nucleic acid binding, etc.) are described in U.S. Pat.Nos. 5,559,410, 5,585,639, 5,576,220, and 5,541,061.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configuarablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols the various high throughput. Thus,for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

III) Assay Components.

A) Polymerized Microtubules

As indicated above, the assays of this invention utilize polymerizedmicrotubules, which, in the presence of a depolymerizing protein orsevering protein are depolymerized or cleaved to produce tubulinmonomers or oligomers. When the depolymerizing or severing proteins areinhibited, the formation of tubulin monomers or oligomers is inhibited.

Virtually any microtubules can be used for the assays of this invention.Means of obtaining such microtubules are well known to those of skill inthe art. Tubulin is available commercially or can be isolated from awide variety of sources, e.g., plants, animal tissues, oocytes, etc. Forexample, tubulin can be isolated from Arabidopsis cells in stationaryphase (day 10 to 11) cultural cells (200 to 500 gm fresh weight) byDEAE-Sephadex A50 chromatography as described by Morejohn et al. (1985).Cell Biol. Int. Rep. 9(9): 849-857 with modifications described inBokros et al. (1993), Biochemistry 32(13): 3437-3447. Briefly,Arabidopsis cells are homogenized in an isolation buffer (IB) consistingof 50 mM PIPES-KOH, pH 6.9, 1 mM EGTA, 0.5 mM MgSO₄, 1 mM DTT and 0.1 mMGTP, supplemented with 50 mg/mL Na-p-tosyl-L-arginine methyl ester(TAME), and 5 mg/mL each of pepstatin A, leupeptin hemisulfate, andaprotinin. The cell homogenate is subjected to DEAE-Sephadex A50chromatography for tubulin isolation. Ammonium sulfate precipitates ofDEAE-isolated tubulin can be aliquoted and stored at −80° C. until use.

Microtubules can be purified to homogeneity by a single taxol-inducedmicrotubule polymerization step in IB supplemented with 1 mM GTP asdescribed previously (Bokros et al. (1993), Biochemistry 32(13):3437-47). Briefly, samples of DEAE-isolated tubulin are thawed andresuspended in IB supplemented with 1 mM DTT and 1 mM GTP, and clarifiedby centrifugation for 1 hr at 100,000×g (2° C.) in a Beckman TL-100ultracentrifuge (TLA-100 rotor). Clarified tubulin is polymerized with atwofold molar excess of taxol in a microtubule assembly buffer composedof IB, 1 mM DTT, 1 mM GTP and 1% DMSO. Assembly of microtubules wasperformed by gradual temperature ramping from 2° C. to 25° C. over a2-hour period. Polymer is collected by centrifugation for 45 min at30,000×g at 25° C. through a cushion of 20% (w/v) sucrose in assemblybuffer.

In a preferred embodiment, the tubulin/microtubules are isolated from ananimal tissue e.g. brain tissue according to the methods of Hyman et al.(1991) Meth Enzy., 196: 478-485. The brain tubulin can be modified withtetramethylrhodamine or fluorescein N-hydroxysuccinimide ester(Molecualr Probes, Inc., Eugene, Oreg.) as described by Hyman et al.(1991) supra.

Most microtubules are in a state of flux, undergoing assembly anddisassembly. In a preferred embodiment of the assays of this inventionmicrotubules are utilized that are stabilized as essentially intactmicrotubules. This can be accomplished by using microtubule4s that arenaturally stable (e.g., axonemal microtubules) or by treating themicrotubules so that they are stabilized. Methods of stabilizingmicrotubules are well known to those of skill in the art and include,but are not limited to the use of stabilizing agents such as paclitaxeland paclitaxel derivatives (e.g., taxotere), non-hydrolyzable nucleotide(e.g., GTP) analogues (e.g., guanylyl-(α,β)-methylene diphosphate(GMPCPP)), and the like.

In a preferred embodiment, taxol-stabilized microtubules are prepared bypolymerizing tubulin (2-10 mg/ml) at 37° C. for 45 minutes in BRB80(80-mM PIPES[pH 6.8], 1 mM MgCl₂, 1 mM EGTA) containing 1 mM GTP and10-% dimethyl sulfoxide (DMSO). Taxol is then added to a concentrationof 20:M.

GMPCPP-stabilized microtubules are prepared by incubating tubulin in theabove, buffer, substituting 0.5 mM GMPCPP for GTP. The GMPCPPmicrotubules can be stored at room temperature without taxol and arepreferably used within 1 day after preparation.

B) Assay Reaction Mixture.

The assays of this invention are performed in a reaction mixture thatprovides the components necessary for microtubule depolymerizing ormicrotubule severing activity of the subject protein (e.g., katanin,XKCM1, OP18 etc.) and that are compatible with the enzymatic activity ofthe subject proteins. Typically the reaction mixture comprises anappropriate buffer (e.g., HEPES pH 6.5-8.0) and an energy supplyingmolecule such as guanosine triphosphate (GTP) for microtubuledepolymerizing proteins or adenosine triphosphate (ATP) for severingmolecules such as katanin. One preferred assay mixture is described inExample I.

C) Microtubule Depolymerizing and Microtubule Severing Agents.

As indicated above, the assays of this invention essentially detect theactivity of a test agent on a microtubule depolymerizing or microtubulesevering polypeptide. Microtubule depolymerizing polypeptides such asOP18 (Belmont et al. (1990) Cell, 62: 579-589) and XKCM1 (Walczak et al.(1996) Cell, 84: 37-47) increase the frequency of catastrophes(transitions of a microtubule from a growing to a shrinking state) andthus promote disassembly of microtubules from their ends.

In contrast to microtubule depolymerizing proteins, other proteins, suchas katanin, promote the disassembly of microtubules by generatinginternal breaks within a microtubule and are referred to as microtubulesevering proteins (see, e.g., Vale (1991) Cell 64: 827-839; Shiina etal. (1994) Science 266: 282-285; Shiina et al. (1992) EMBO J. 11:4723-4731; McNally and Vale (1993) Cell, 75: 419-429).

Preferred microtubule depolymerizing proteins for the methods of thisinvention include, but are not limited to XKCM 1, and OP18, whilepreferred microtubule severing proteins include katanin.

i) Katanin.

Katanin, a heterodimer of 60 kDa and 80 kDa subunits purified from seaurchin eggs, is unique among the known microtubule and actin severingproteins in that it disrupts contacts within the polymer lattice byusing energy derived from ATP hydrolysis (McNally and Vale (1993) Cell,75: 419-429). Katanin acts substoichiometrically, as one molecule ofkatanin can release several tubulin dimers from a microtubule. Katanindoes not appear to proteolyze or modify tubulin, since the tubulinreleased from the disassembly reaction is capable of repolymerizing(McNally and Vale (1993) Cell, 75: 419-429). The mechanism ofmicrotubule severing by katanin, however, is not understood.

Katanin-catalyzed microtubule severing and disassembly could potentiallybe involved in several changes in the microtubule cytoskeleton observedin vivo. Recent studies have shown that katanin is concentrated at thecentrosome in a microtubule-dependent manner in sea urchin embryos(McNally et al. (1996) J. Cell Sci. 109: 561-567). One phenomenon thatcould require disassembly of microtubules at the centrosome is thepoleward flux of tubulin in the mitotic spindle (Mitchison (1989) J.Cell Biol. 109: 637-652). The disassembly of microtubule minus ends atthe spindle pole during poleward flux could be driven by katanin, orkatanin could simply allow depolymerization by uncapping microtubuleminus ends that are docked onto γ-tubulin ring complexes (Zheng et al.(1995) Nature 378: 578-583; Moritz et al. (1995) Nature 378: 638-640).Another possible role for katanin at the centrosome is in promoting therelease of microtubules from their centrosomal attachment points.Microtubules are nucleated from γ-tubulin ring complexes at thecentrosome (Joshi et al., 1992; Moritz et al. (1995) Nature 378:638-640), but release of microtubule minus ends has been observedindirectly in Dictyostelium (Kitanishi-Yumura et al. (1987) Cell Motil.Cytoskeleton 8: 106-117) and directly in PtKl cells (Keating (1997)Proc. Natl. Acad. Sci. USA, 94: 5078-5083) and Xenopus egg extracts(Belmont et al. (1990) Cell 62: 579-589). Finally, katanin couldaccelerate the rapid disassembly of the interphase microtubule networkat the G2/M transition (Zhai et al. (1996) J. Cell Biol. 135: 201-214)by severing cytoplasmic microtubules, which would increase the number offree microtubule ends from which depolymerization could occur.Regardless of the particular mode of activity, modulation of kataninactivity will have profound effects on the cell cycle.

The amino acid and nucleic acid sequences of the P60 and P80 subunits ofkatanin are provided in FIG. 1 and (see also SEQ ID NO: 1 and SEQ ID NO:2). It was a discovery of this invention that the microtubule severingactivity resides entirely in the P60 subunit. Thus the assays of thisinvention can be practiced either with the heterodimeric katanin or witha P60 subunit alone.

The P60 and/or P80 subunits of katanin can be purified (e.g., from seaurchin eggs, e.g., eggs from Strongylocentrotus purpuratus) as describedby McNally and Vale (1993) Cell, 75: 419-429. Alternatively, either orboth subunits can be recombinantly expressed and purified as describedbelow and in Example 1.

ii) XKCM1

XKCM1 (for Xenopus kinesin central motor 1) is a motor protein essentialfor mitotic spindle assembly in vitro. XKCM1 localizes to centromeresand appears to regulate the polymerization dynamics of microtubules. Theisolation of an XKCM1 clone is described by Walczak et al (1996) Cell,84: 37-47, and a nucleic acid sequence of an XKCM1 cDNA is providedtherein and in SEQ ID NO: 3). Using this sequence information, XKCM1 canbe expressed as described below and by Walczak et al. (1996) supra.

iii) OP18

Another microtubule depolymerizing motor protein suitable for use in themethods of this invention is OP18, also called stathmin orstathmin/op18. OP18 is described in detail by Gradin et al. (1998) J.Cell Biol., 140(1):131-141, by Andersen et al. (1997) Nature,389(6651):640-643, by Larsson et al. (1997) Mol. Cell. Biol.,17(9):5530-5539, and by Belmont et al. (1996) Cell, 84(4):623-631.

iv) Other Microtubule Severing or Depolymerizing Proteins.

Other microtubule depolymerizing or severing proteins include, but arenot limited to elongation factor-1α (Shiina et al. (1994) Science 266:282-285) and a novel homo-oligomeric protein described by Shiina et al.(1992) EMBO J. 11: 4723-4731.

Other microtubule depolymerizing or severing proteins can be identifiedwith only routine experimentation. The assays used to identifymicrotubule depolymerizing or severing proteins are identical to theassays described herein, the only difference being that no test agent isrequired. A detailed example of the assay of a microtuble severingprotein (katanin) is provided in McNally and Vale (1993) Cell, 75:419-429. The same approach can readily be used to identify othersevering or depolymerizing proteins.

IV) Detection Methods.

Any detection method that allows detection and/or quantification of theamount or rate of appearance of tubulin monomers or oligomers and/or therate of disappearance of assembled (polymerized) microtubules can beused in the assays of this invention. Preferred detection methodsinclude, but are not limited to video microscopy; DAPI fluorescencechanges, fluorescence resonance energy transfer and centrifugation.

A) Video Microscopy.

In one embodiment, microtubule depolymerization or severing is detectedby microscopy (visually or using a video or photographic recordingdevice). Assays involving microscopic visualization of microtubulespreferably utilize labeled (e.g., fluorescently labeled) microtubules.The Microtubules are preferably immobilized on a solid support (e.g., aglass slide), and exposed to a solution containing the microtubuledepolymerizing or severing protein and an a nucleoside triphosphate. Theintact and depolymerized or severed microtubules can be directlyvisualized using a microscope. Microtubule depolymerization in thecontrol and the assay containing the test agent can be visualized sideby side or sequentially.

The microscope can optionally be equipped with a still camera or a videocamera and may be equipped with image acquisition and analysis softwareto quantify the relative abundance of intact and fragmentedmicrotubules.

This method can be used with essentially any label that can bevisualized in a microscope. Such labels include, but are not limited tofluorescent labels (e.g., fluorescein, rhodamine, etc.), colorimetriclabels, and radioactive labels (with appropriate scintillation screen),and the like. In some embodiments of the assays of this invention, themicrotubules can be visualized without any label (e.g. via differentialinterference contrast microscopy).

An illustration of the use of video microscopy to visualize microtubulesevering by katanin is provided by Mcnally et al. (1993) Cell, 75:419-429. In this case, the microtubules are labeled with rhodamine andthe images of the severed microtubules are captured digitally.

B) DAPI Fluorescence Changes.

In another embodiment, the state of microtubule polymerization can bedetermined by changes in fluorescence of DAPI stained microtubules. Ithas been shown that DAPI fluorescence intensity is higher when this dyeis bound to polymerized versus free tubulin (Heusele et al. (1987) Eur.J. Biochem. 165: 613-620). When katanin and ATP were incubated withDAPI-labeled microtubules, a linear decrease in fluorescence intensityis observed as a function of time, reflecting the conversion ofmicrotubules to tubulin.

Assay can thus be performed as described above with DAPI labeledstabilized microtubules. The rate or amount decrease in DAPIfluorescence is detected as described by Heusele et al. supra. Thechange in fluorescence with a test agent is compared to that observed ina negative and/or positive control reaction.

It was a surprising discovery of this invention that tubulin, tubulindimers, tubulin oligomers or microtubules can be labeled with variouslabels such as DAPI and that the label does not interfere with theinteraction of various test agents or cytoskeletal associated proteinswith the labeled tubulin to a degree that would prevent assaying theimpact of a test agent on microtubule polymerization, and/ordepolymerization, and/or severing. Labels that can be used include, butare not limited to anilinonapthalene sulfonate (ANS) (e.g. MolecularProbes Catalogue Nos: A-47, A-50, T-53, etc.), bis-ANS (Molecular ProbesCatalogue No: B-153), N-phenyl-1-naphthylene (NPN) (Molecular ProbesCatalogue No: P-65), DCVJ (Molecular Probes Catalogue No: D-3923),ruthenium red, and cresol violet.

C) Fluorescence Resonance Energy Transfer

The degree of microtubule polymerization/depolymerization can also bedetermined by fluorescent resonance energy transfer (FRET). Fluorescenceresonance energy transfer, a phenomenon that occurs when twofluorophores with overlapping absorption and emission spectra arelocated close together (e.g., <7 nm) apart (Stryer (1978) Ann. Rev.Biochem., 47: 819-846. FRET is a powerful technique for measuringprotein-protein associations and has been used previously tomeasure thepolymerization of monomeric actin into a polymer (Taylor et al. (1981)J. Cell Biol., 89: 362-367) and actin filament disassembly by severing(Yamamoto et al. (1982) J. Cell Biol., 95: 711-719).

In a preferred embodiment, equimolar proportions of differently labeled(e.g., fluorescein labeled and rhoadmine-labeled) tubulin are combined.The fluorescence is quenched upon tubulin polymerization indicating thatthe tubulin-bound fluorochromes in a microtubule come in close enoughproximity for energy transfer to occur. When the microtubule isdepolymerized or severed, a rapid unquenching of (e.g. fluorescein)fluorescence is observed.

The rates and/or amount of fluorescence generated by a reaction with atest agent and a control can be compared. A decrease in rate or amountof fluorescence in the presence of a test agent indicates inhibitoryactivity on the microtubule depolymerizing or severing protein(s).

In particularly preferred embodiment, the microtubules are polymerizedfrom a mixture of equal concentrations of fluorescein and rhodaminetubulin and diluted to 600:g/ml tubulin in BRB80 containing 20:M taxoland the oxygen-depleting system consisting of glucose oxidase (30:g/ml),catalase (100 mg/ml), glucose (10 mM), and dithiothreitol (10 mM)(Kishino and Yanigida (1988) nature 334: 74-76). Aliquots (150:1) ofthese microtubules can be mixed e.g., with samples of purified severingprotein and the fluorescence from the fluorescein (excitation 492 nm;emission 518 nm) is recorded. The reaction is preferably run with apositive and negative control. A detailed FRET assay for tubulinpolymerization is found in McNally et al. (1993) Cell, 75: 419-429.

D) Centrifugation.

microtubule disassembly in solution can be documented in a quantitativemanner by examining the relative amounts of sedimentable andnonsedimentable tubulin after incubation with the severing ordepolymerizing protein (e.g. katanin) and ATP or GTP. In the absence ofa modulating agent, when taxol-stabilized microtubules are incubatedtithe the katanin p80 and p60 subunits and ATP, nondsedimentable tubulinis released from microtubule polymer in an approximately linear manner.The rate of release varies with microtubule depolymerizing or severingprotein concentration and will be dependent on the activity of amodulating “test” agent if present.

In a preferred embodiment, fluorescent microtubules (e.g., 100-300:g/mltubulin) are incubated with the microtubule depolymerizing or severingprotein in buffer (e.g. 20 mM HEPES (pH 7.5), 2 mM MgCl2, 25 mMK-glutamate, 0.02% Triton X-100, 250:g/ml SBTI, and 20:M taxol or taxolderivative), at various times. Aliquots e.g. of 100:1 are brought to 10mM ADP or GDP (to stop the severing or depolymerizing reaction) andsedimented e.g., at 228,000×g for 10 minutes. Supernatants are removed,and pellets are resuspended in 100:L of buffer. The pellets andsupernatants can be brought up to 300:L e.g. with BRB80, and therelative fluorescence signals in the supernatant and the pellet arequantitated using a Perkin Elmer L25B luminescence spectromoter.

E) Liquid Crystal Assay Systems.

In still another embodiment, binding of the microtubule depolymerizingor severing proteins to microtubules can be detected by the use ofliquid crystals. Alternatively, it is expected that liquid crystals canbe used to monitor the state of tubulin polymerization.

Liquid crystals can be used to amplify and transduce receptor-mediatedbinding of proteins at surfaces into optical outputs. Spontaneouslyorganized surfaces can be designed so that protein molecules, uponbinding to ligands (e.g. microtubules) hosted on these surfaces, triggerchanges in the orientations of 1- to 20-micrometer-thick films ofsupported liquid crystals, thus corresponding to a reorientation of ˜10⁵to 10⁶ mesogens per protein. Binding-induced changes in the intensity oflight transmitted through the liquid crystal are easily seen with thenaked eye and can be further amplified by using surfaces designed sothat protein-ligand recognition causes twisted nematic liquid crystalsto untwist (see, e.g., Gupta et al. (1998) Science, 279: 2077-2080).This approach to the detection of ligand-receptor binding does notrequire labeling of the analyte, does not require the use ofelectroanalytical apparatus, provides a spatial resolution ofmicrometers, and is sufficiently simple that it is useful in biochemicalassays and imaging of spatially resolved chemical libraries.

D) Synthesis or Expression of Microtubule Depolymerizing or ServingProteins.

i) De novo Chemical Synthesis.

Using the information provided herein, the microtubule depolymerizing orsevering proteins, protein subunits, or subsequences thereof may besynthesized using standard chemical peptide synthesis techniques. Wherethe desired subsequences are relatively short the molecule may besynthesized as a single contiguous polypeptide. Where larger moleculesare desired, subsequences can be synthesized separately (in one or moreunits) and then fused by condensation of the amino terminus of onemolecule with the carboxyl terminus of the other molecule therebyforming a peptide bond.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, andStewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill.

ii) Recombinant Expression.

In a preferred embodiment, the microtubule depolymerizing or severingproteins, protein subunits, or subsequences, are synthesized usingrecombinant DNA methodology. Generally this involves creating a DNAsequence that encodes the protein, placing the DNA in an expressioncassette under the control of a particular promoter, expressing theprotein in a host, isolating the expressed protein and, if required,renaturing the protein.

DNA encoding the microtubule depolymerizing or severing proteins,protein subunits, or subsequences of this invention can be prepared byany suitable method as described above, including, for example, cloningand restriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. (1979) Meth.Enzymol. 68: 90-99; the phosphodiester method of Brown et al.(1979)Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method ofBeaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solidsupport method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce he desired DNA sequence.

In one embodiment, the microtubule depolymerizing or severing proteins,of this invention can be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the nucleic acidsequence or subsequence is PCR amplified, using a sense primercontaining one restriction site (e.g., NdeI) and an antisense primercontaining another restriction site (e.g., HindIII). This will produce anucleic acid encoding the desired the microtubule depolymerizing orsevering protein having terminal restriction sites. This nucleic acidcan then be easily ligated into a vector containing a nucleic acidencoding the second molecule and having the appropriate correspondingrestriction sites.

Suitable PCR primers can be determined by one of skill in the art usingthe sequence information provided herein. Appropriate restriction sitescan also be added to the nucleic acid encoding the microtubuledepolymerizing or severing proteins by site-directed mutagenesis. Theplasmid containing the microtubule depolymerizing or severingprotein-encoding nucleic acid is cleaved with the appropriaterestriction endonuclease and then ligated into the vector encoding thesecond molecule according to standard methods.

The nucleic acid sequences encoding the microtubule depolymerizing orsevering proteins may be expressed in a variety of host cells, includingE. coli, other bacterial hosts, yeast, and various higher eukaryoticcells such as the COS, CHO and HeLa cells lines and myeloma cell lines.As the microtubule depolymerizing or severing proteins are typicallyfound in eukaryotes, a eukaryote host is preferred. The recombinantprotein gene will be operably linked to appropriate expression controlsequences for each host. For E. coli this includes a promoter such asthe T7, trp, or lambda promoters, a ribosome binding site and preferablya transcription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant the microtubule depolymerizing orsevering proteins can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like (see, generally,R. Scopes, (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher(1990) Methods in Enzymology Vol. 182: Guide to Protein Purification.,Academic Press, Inc. N.Y.). Substantially pure compositions of at leastabout 90 to 95% homogeneity are preferred, and 98 to 99% or morehomogeneity are most preferred. Once purified, partially or tohomogeneity as desired, the polypeptides may then be used (e.g., asimmunogens for antibody production).

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the microtubule depolymerizingor severing protein (s) may possess a conformation substantiallydifferent than the native conformations of the constituent polypeptides.In this case, it may be necessary to denature and reduce the polypeptideand then to cause the polypeptide to re-fold into the preferredconformation. Methods of reducing and denaturing proteins and inducingre-folding are well known to those of skill in the art (see, Debinski etal. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993)Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski et al., for example, describes thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The protein is then refolded in a redox buffer containing oxidizedglutathione and L-arginine.

One of skill would recognize that modifications can be made to themicrotubule depolymerizing or severing proteins without diminishingtheir biological activity. Some modifications may be made to facilitatethe cloning, expression, or incorporation of the targeting molecule intoa fusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids (e.g.,poly His) placed on either terminus to create conveniently locatedrestriction sites or termination codons or purification sequences.

In a particularly preferred embodiment, the katanin protein(s) areexpressed as described in Example 1, while XKCM1 is expressed andpurified as described by Walczak et al. (1996) supra.

IV. Data Management.

In one embodiment, the assays of this invention are facilitated by theuse of databases to record assay results. Particular with the use oflarge-scale screening systems, (e.g., screening of combinatoriallibraries) data management can become a significant issue. For example,all natural hexapeptides have been synthesized in a single combinatorialexperiment producing about 64 million different molecules. Maintenanceand management of even a small fraction of the information obtained byscreening such a library is aided by methods automated informationretrieval, e.g. a computer database.

Such a database is useful for a variety of functions, including, but notlimited to library registration, library or result display, libraryand/or result specification, documentation, and data retrieval andexploratory data analysis. The registration function of a databaseprovides recordation/registration of combinatorial mixtures and assayresults to protect proprietary information in a manner analogous to theregistration/protection of tangible proprietary substances. Library andassay result display functions provide an effective means to reviewand/or categorize relevant assay data. Where the assays utilize complexcombinatorial mixtures for test agents, the database is useful forlibrary specification/description. The database also providesdocumentation of assay results and the ability to rapidly retrieve,correlate (or other statistical analysis), and evaluate assay data.

Thus, in some preferred embodiments, the assays of this inventionadditionally involve entering test agent(s) identified as positive(i.e., having an effect on microtubule polymerization, and/ordepolymerization, and/or severing) in a database of “positive” compoundsand more preferably in a database of therapeutic or bioagricultural leadcompounds.

The database can be any medium convenient for recording and retrievinginformation generated by the assays of this invention. Such databasesinclude, but are not limited to manual recordation and indexing systems(e.g. file-card indexing systems). However, the databases are mostuseful when the data therein can be easily and rapidly retrieved andmanipulated (e.g. sorted, classified, analyzed, and/or otherwiseorganized). Thus, in a preferred embodiment, the signature the databasesof this invention are most preferably “automated”, e.g., electronic(e.g. computer-based) databases. The database can be present on anindividual “stand-alone” computer system, or a component of ordistributed across multiple “nodes” (processors) on a distributedcomputer systems. Computer systems for use in storage and manipulationof databases are well known to those of skill in the art and include,but are not limited to “personal computer systems”, mainframe systems,distributed nodes on an inter- or intra-net, data or databases stored inspecialized hardware (e.g. in microchips), and the like.

III. Kits for Screening for Modulators of Microtubule Depolymerizing orMicrotubule Severing Agents.

In still another embodiment, this invention provides kits for thepractice of any of the methods described herein. The kits comprise oneor more containers containing one or more of the assay componentsdescribed herein. Such components include, but are not limited tostabilized microtubules, microtubule depolymerizing or microtubulesevering proteins or protein subunits, one or more test agents, reactionmedia, solid supports (e.g., microtitre plates) with attachedcomponents, buffers, labels, and other reagents as described herein.

The kits may optionally include instructional materials containingdirections (i.e., protocols) for carrying out any of the assaysdescribed herein. While the instructional materials typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Katanin, a Microtubule-severing Protein is a Novel AAA ATPaseThat Targets to the Centrosome Using a WD40-Containing Subunit RESULTS

To begin dissecting the functional domains of katanin, we isolated cDNAclones for the p60 and p80 subunits from cDNA derived from sea urchin(Strongylocentrotus purpuratus) egg mRNA. After first obtaining peptidesequence of several proteolytic fragments from the two sea urchinkatanin subunits, cDNA clones were isolated using a combination ofdegenerate PCR, cDNA library screening, and anchor-ligated PCR (seeExperimental Procedures). The predicted amino acid sequences of the cDNAclones contained 139 amino acids (a.a.) and 306 amino acids of peptidesequences obtained by direct microsequencing of p60 and p80respectively.

p60 is a Novel Member of the AAA ATPase Superfamily

Sequence analysis of the p60 cDNA clone revealed an open reading framethat encodes a 516 a.a. polypeptide (FIG. 1A). A BLAST search with thepredicted p60 protein sequence revealed that this polypeptide contains aC-terminal domain (a.a. 231-447) that is highly conserved in the AAAATPase superfamily (FIG. 1B) (Confalonieri et al. (1995) BioEssays 17:639-650). This ˜220 amino acid region contains the “Walker A” (P-loop)and “Walker B” motifs found in many ATPases (Walker et al. (1982) EMBOJ. 1: 945-951). AAA proteins, which contain either one or two of these220 a.a. ATP-binding modules, constitute a large superfamily whosemembers have been implicated in a variety of cellular functions(Confalonieri et al. (1995) supra.).

Of the AAA domains entered into sequence data bases, mei-1, a C. elegansprotein required for meiosis (Clark-Maguire et al. (1994) Genetics 136:533-546), is most closely related to p60 (55% a.a. identify, FIG. 1B).Mei-1 was discovered in a genetic screen as a protein that is requiredfor meiotic spindle formation, but disappears during subsequent mitoticdivisions. Interestingly, both p60 (McNally et al. (1996) J. Cell Sci.109: 561-567) and mei-l (Clark-Maguire et al. (1994) J. Cell Biol. 126:199-209) are localized to spindle poles in a microtubule-dependentmanner. However, the N-terminal half of p60 has no significant homologyto mei-1, suggesting that p60 and mei-1 may not be orthologs. BLASTsearches with p60 sequences, however, revealed several human ESTs(expressed sequence tags) that have strong amino acid identity outsideof the AAA domain, suggesting the existence of vertebrate homologs ofp60.

p80 Contains WD40 Repeats

Sequence analysis of the sea urchin p80 cDNA clone revealed a predicted690 a.a. polypeptide that contains six “WD40” repeat motifs extendingfrom residues 1-256 (FIG. 2A). An alignment of these repeats with twounrelated WD40 repeat-containing proteins is shown in FIG. 2B. The WD40repeats in several proteins have been documented to participate inprotein-protein binding interactions (Komachi et al. (1994) Genes Dev.8: 2857-2867; Wall et al. (1995) Cell 83: 1047-1058). The C-terminalregion of p80 (residues 257-690) did not exhibit significant amino acididentity to any previously described protein. However, significantidentity of sea urchin p80 was observed with several human EST clones.The sequences of these clones were used to isolate a full-length humanp80 katanin homolog by PCR (see Experimental Procedures). The human cDNAencodes a predicted 655 a.a. protein with 61% a.a. identity in the WD40domain (a.a. 1-256) (FIG. 2B), 23% a.a. identity in the central 187residues, and 54% a.a. identity in the C-terminal 164 a.a. with S.purpuratus p80 katanin (latter two regions are not shown).

Baculovirus Expression and Molecular Structure of the Katanin Subunits

Deciphering the roles of the two katanin subunits is essential forunderstanding the enzyme's mechanism and biological activities. However,separation of the native sea urchin p60/p80 subunits requires denaturingconditions. We therefore sought to express the two subunits together andseparately and then test their enzymatic activities. Bacterialexpression of p60 produced largely insoluble protein, and the smallamount of soluble p60 had no microtubule-stimulated ATPase activity(data not shown). However, using the baculovirus expression system, weobtained soluble p60, p80, and the p60/p80 complex (each expressed witha N-terminal His₍₆₎ tag), and purified the expressed proteins usingmetal affinity chromatography (FIG. 3A). When p60 and p80 wereco-expressed, the stoichiometry of the two subunits in the purifiedprotein was approximately equal (1.0:0.9 p60:p80 molar ratio, asdetermined by Coomassie staining). Moreover, immunoprecipitation with ananti-p60 antibody led to co-immunoprecipitation of equal quantities ofp60 and p80 (FIG. 3B). These results indicate that baculovirus-expressedp60 and p80 heterodimerize, as observed with native katanin (McNally andVale (1993) Cell, 75: 419-429).

To examine katanin's structure, baculovirus-expressed p60, p80, orp60/80 was adsorbed onto mica chips, and the chips were subsequentlyfrozen, etched, and rotary shadowed with platinum (Heuser (1989) J.Electron Microsc. Technique 13:, 244-263; Heuser (1983) J. Mol. Biol.169: 155-195). The platinum-shadowed p60 appeared as a 14-16 nm ringpunctuated in the center by a 3-5 nm opening, often with what appears tobe cracks radiating outward (FIG. 4A). p80, on the other hand, appearedas ˜11 nm particles and occasional unstructured protein aggregates;rings were not observed (FIG. 4B). Rings were also seen for p60/p80complexes (FIGS. 4C, 4D) and native sea urchin katanin (data not shown).Interestingly, two types of p60/p80 complexes were visible: large ˜20 nmdiameter rings with bright edges, which is suggestive of tallercomplexes that extend upward from the mica (FIG. 4D), and smaller ringsof the size of p60 alone with several p80 sized particles radiating fromthe central ring (FIG. 4C). The large and small rings might representclosed and “splayed” versions of the p60/p80 complex, respectively,which could be produced if the complex dissociates upon mica adsorption.Both p60 and p60/p80 structures resemble the rings observed for the AAAATPases NSF and p⁹⁷, whose dimensions are 15-17 nm (Hanson el al. (1997)Cell 90: 523-535).

p60 Katanin has Microtubule-Stimulated ATPase and Severing Activity

With the availability of isolated p60 and p80, we then examined whetherthe individual subunits have ATPase activity. The co-expressed p60/p80heterodimer displayed an ATP turnover rate of 0.3 ATP/sec/heterodimer;this activity was stimulated ˜10-fold by microtubules (FIG. 5A). Thisbasal activity and the fold stimulation by microtubules are similar tothat observed for native sea urchin katanin (data not shown). Consistentwith the finding of an AAA domain in its sequence, p60 alone displayed amicrotubule-stimulated ATPase activity. Surprisingly, the maximal basaland microtubule-stimulated ATPase rates of p60 were only 2-fold lowerthan those of the p60/p80 heterodimer (FIG. 5A). p80 itself had nodetectable ATPase activity. The activation of ATPase activity bymicrotubules displayed an atypical, non-hyperbolic behavior. ATPturnover by p60 and p60/p80 was stimulated at low concentrations ofmicrotubules (peak at ˜2 μM tubulin), but then decreased at highermicrotubule concentrations (FIG. 5A). This same complex pattern ofmicrotubule stimulation was also observed for native sea urchin katanin(data not shown).

We then tested the microtubule severing activity of p60, p80, andp60/p80 using a fluorescence microscopy assay (McNally and Vale (1993)Cell, 75: 419-429). Both p60 and p60/p80 severed microtubules in thisassay (FIG. 5B). Broken microtubules were observed within 1 min afterintroducing 0.1 μM p60 or p60/p80, and microtubules were completelydisassembled after 5 min. The reaction appeared somewhat slower with p60alone. Microtubules remained intact if ATP was omitted from the reaction(not shown). In contrast, p80 was unable to sever microtubules atconcentrations 5-fold higher than those used for p60 (FIG. 5B). Theseexperiments demonstrate that p60 alone can carry out all of the stepsnecessary for coupling ATP hydrolysis to microtubule disassembly.

To better compare the microtubule severing activities of p60 andp60/p80, we developed a quantitative microtubule disassembly assay basedupon a previous finding that DAPI fluorescence intensity is higher whenthis dye is bound to polymerized versus free tubulin (Heusele et al.(1987) Eur. J. Biochem. 165: 613-620). When katanin and ATP wereincubated with DAPI-labeled microtubules, a linear decrease influorescence intensity was observed as a function of time, reflectingthe conversion of microtubules to tubulin (FIG. 5C). The loss ofmicrotubule polymer was confirmed by centrifugation studies, whichshowed an increase in non-sedimentable tubulin with a similar timecourse (data not shown). The fluorescence decrease induced by theseenzymes reached a steady-state level that was slightly higher than pure,monomeric tubulin, suggesting that some tubulin oligomer may exist atsteady state. The rate of fluorescence decrease was proportional to p60or p60/p80 concentration over a 10-fold range (data not shown). When therates of microtubule disassembly were compared, p60 was half as activeas p60/p80 (FIG. 5C). This slower rate of microtubule disassembly isconsistent with the previously described 2-fold decrease in ATPaseactivity of p60 compared with p60/p80.

The p80 WD40 Domain Targets to the Centrosome

The finding that p60 by itself can sever microtubules left open thequestion of the function of the p80 katanin subunit. At least twofunctional domains of p80 could be postulated. First, since katanin is aheterodimer (McNally and Vale (1993) Cell, 75: 419-429), some part ofp80 must be involved in heterodimerization with p60. Second, becauseprevious studies have shown that katanin is concentrated at centrosomesin vivo (McNally et al. (1996) J. Cell Sci. 109: 561-567), p80 couldcontain a domain that interacts with a centrosomal protein to allowtargeting of the katanin holoenzyme. Because WD40 repeats have beenimplicated in heterophilic protein-protein interactions (Komachi et al.(1994) Genes Dev. 8: 2857-2867; Wall et al. (1995) Cell 83: 1047-1058),the six WD40 repeats in p80 represented a good candidate domain forparticipating in either dimerization or centrosome targeting.

In order to test whether the WD40 repeats of p80 are required forheterodimerization with p60, we deleted the entire WD40 domain andexamined whether the truncated p80 (p80□1-302) interacted with p60 whenthe two polypeptides were co-translated in a rabbit reticulocyte system.The truncated p80 (p88Δ1-302) was co-immunoprecipitated by the anti-p60antibody only in the presence of p60 and just as efficiently as fulllength p80 (FIG. 6). This finding indicates that the WD40 repeats arenot required for dimerizing the two katanin subunits. Nevertheless, itremained possible that the WD40 domain was one of multiple, redundantp60-interacting domains. However, a C-terminal truncation of p80(p80Δ303-690) containing only the WD40 domain did notco-immunoprecipitate with p60 (FIG. 6). These results indicate that thep80 WD40 repeats are neither necessary nor sufficient for dimerizationwith p60. To determine which region of p80is required for interactionwith p60, a p80 deletion lacking the C-terminal 130 amino acids(p80Δ560-690) was constructed and was found not to co-immunoprecipitatewith p60 (FIG. 6). These findings suggest that the C-terminal 130 aminoacids of p80, but not the WD40 repeat domain, are involved in thedimerization with p60.

To examine whether the p80 WD40 repeats bind to a protein in thecentrosome, we tested whether these repeats can target a heterologousprotein (green fluorescent protein, GFP) to the centrosome aftertransient transfection in the human fibroblast cell line, MSU1.1 (Lin etal. (1995) Int. J. Cancer 63: 140-147). The WD40 domain of human p80katanin was used, because it was more likely that the human proteinwould interact with centrosomal proteins in this human cell line.

Immunofluorescence of MSU1.1 cells with an antibody specific for humanp80 katanin (see Experimental Procedures) showed labeling of thecytoplasm and more concentrated staining at one or two spots thatco-localized with γ-tubulin staining (FIG. 7), confirming thatendogenous katanin is concentrated at centrosomes in fibroblasts as itis in sea urchin embryos (McNally et al. (1996) J. Cell Sci. 109:561-567). In contrast to the localization in sea urchin embryos, theconcentration of p80 at centrosomes in fibroblasts remained aftercomplete depolymerization of microtubules with nocodazole (data notshown), suggesting that katanin is bound to the pericentriolar material.When a fusion protein consisting of the six WD40 repeats of human p80katanin (a.a. 1-263) appended to the N-terminus of green fluorescentprotein (GFP) was expressed in MSU1.1 cells, one or two foci of greenfluorescence that co-localized with γ-tubulin staining was observed 2-4hr after transfection in addition to diffuse cytoplasmic fluorescence(FIG. 7). Identical results were obtained in transfections of Hela cells(not shown). In contrast to these findings, cells transfected with GFPalone never revealed foci of green fluorescence at centrosomes (notshown). After longer periods of expression (8-24 hr) of p80 WD40-GFP,numerous heterogeneously-sized bright foci of green fluorescenceappeared that did not co-localize with γ-tubulin, and later, massiveaggregates several □m in diameter were observed (not shown). Theseresults indicate that the WD40 repeats of human p80 katanin aresufficient to target GFP to the centrosome and suggest that once thecentrosome binding sites are saturated, the additional fusion proteinaggregates in the cytoplasm.

DISCUSSION

Katanin is a unique enzyme that couples ATP hydrolysis to thedissociation of tubulin subunits from the microtubule lattice (McNallyand Vale (1993) Cell, 75: 419-429). Other than the motor proteinskinesin and dynein, katanin is the only known microtubule-associatedATPase. In this study, we have determined the primary structure of thep60 and p80 katanin subunits and examined the roles of the two subunitsin microtubule severing and the cellular localization of the enzyme.

Mechanism of Katanin-Mediated Microtubule Severing

Sequence analysis of p60 katanin revealed that it is a novel member ofthe AAA family of ATPases. This finding suggested that p60 might beresponsible for the previously reported ATPase activity of the nativekatanin dimer (McNally and Vale (1993) Cell, 75: 419-429). However,neither p60 nor p80 contained an identifiable microtubule bindingsequence, such as those found in tau (Butner and Kirschner (1991) J.Cell Biol. 115: 717-730) or MAP1B (Noble et al. (1989) J. Cell Biol.109: 3367-3376), and therefore it was not possible to ascribe themicrotubule binding and severing activities of katanin to either subunitbased upon sequence information alone. By measuring the activities ofthe p60 and p80 subunits purified individually and together as a dimer,we have found that katanin's p60 subunit exhibits bothmicrotubule-stimulated ATPase activity and microtubule-severing activityin the absence of the p80 subunit. Since p60 has all elements requiredfor functional interactions with microtubules, future structure-functionstudies on the mechanism of microtubule severing can be focused on thissingle subunit. Furthermore, we have found that p60 katanin can formrings, the dimensions and appearance of which are similar to thosereported for the AAA proteins NSF and p97 (Hanson et al. (1997) Cell 90:523-535). The comparison of p60 with other AAA proteins provides cluesas to bow katanin disassembles microtubules, as discussed below.

The ATPase properties of katanin show both similarities and differenceswith other AAA family members. Katanin's basal ATPase activity of 0.3ATP/katanin/sec and maximal microtubule-stimulated rate of 3ATP/katanin/sec are comparable to values of 1 ATP/sec for p97 (Peters etal. (1992) J. Mol. Biol., 223: 557-571) and 0.08 ATP/sec for recombinantNSF (Morgan et al. (1994) J. Biol. Chem. 269: 29347-29350). NSF ATPaseis also stimulated two-fold upon binding to its target protein, α- orγ-SNAP (Morgan et al. (1994) J. Biol. Chem. 269: 29347-29350). However,katanin's ATPase activity displays a complex stimulation bymicrotubules. At low microtubule concentrations (<2 μM), ATPase activityincreases with increasing microtubule concentration, but at highermicrotubule concentrations, ATPase activity decreases until iteventually approaches basal levels. In contrast, stimulation of kinesinATPase by microtubules (Gilbert et al. (1993) Biochemistry 32:4677-4684) displays typical hyperbolic curves that reach saturation.

At least two potential explanations could account for the unusual ATPasebehavior of katanin. One possibility is that katanin binds microtubulesat two sites, which could elevate the local microtubule concentration bycrosslinking and thereby stimulate katanin's ATPase activity. At highermicrotubule concentrations, however, the ratio of katanin tomicrotubules would be lower, resulting in a less-crosslinked network andless stimulation of ATPase activity. In support of this idea, bundlingof microtubules by katanin has been observed by microscopy (unpublishedobservations). This behavior has been seen in anothercytoskeletal-polymer stimulated ATPase, Acanthamoeba myosin I, which hastwo discrete actin binding sites: a low affinity catalyic site and ahigher affinity site not involved in catalysis (Lynch et al. (1986) J.Biol. Chem. 261: 17156-17162).

A second explanation for katanin's complex enzymatic behavior couldinvolve katanin oligomerization into rings. Rotary-shadowing EM imagesshow oligomeric ring structures in katanin preparations; howeverhydrodynamic experiments with both native (McNally and Vale (1993) Cell,75: 419-429) and recombinant katanin (data not shown) suggest that themajority of the protein is monomeric. One hypothesis is thatmicrotubules promote p60—p60 oligomerization, and that the assembly ofp60 monomers into a higher order structure on the microtubule stimulatesATPase activity. According to this idea, low microtubule concentrationswould facilitate multimerization, since p60 monomers would be morelikely to bind near one another on the microtubule. High microtubuleconcentrations, on the other hand, would inhibit p60 assembly bysequestering p60 monomers at noncontiguous sites on the lattice. Selfassembly into rings also has been suggested as the cause of dynamin'sbiphasic stimulation of GTPase activity (Tuma and Collins (1994) J.Biol. Chem. 269: 30842-30847; Warnock et al. (1996) J. Biol. Chem. 271:22310-22314). Cryo-electron microscopy studies of the p60-microtubulecomplex provide a means of testing this hypothesis.

Based upon studies of other AAA family members, katanin oligomers/ringsmay prove to be important in the severing mechanism. Although servingdiverse functions, many AAA proteins appear to share a common functionas nucleotide-dependent molecular chaperones that disassemble proteincomplexes (Confalonieri and Duguet (1995) supra.). The best studied AAAmember is NSF, which binds to and induces the disassembly of ternarySNARE complexes after hydrolysis of ATP (Hanson et al. (1995) J. Biol.Chem. 270: 16955-16961; Hayashi et al. (1995) EMBO J. 14: 2317-2325).This reaction plays a role either in vesicle fusion and/or recycling ofcomponents in membrane trafficking pathways. Recently, electronmicroscopy studies have revealed that the NSF ring structure adoptsextended and compact conformations in the ATP-γ-S and ADP states,respectively (Hanson et al. (1997) Cell 90: 523-535). If attached atseveral points to a protein complex, this transition could break apartbonds in the SNARE complex (Hanson et al., (1997) supra.). Katanin maywork in an analogous fashion. A ring of katanin's dimensions couldpotentially contact multiple tubulin sites on a microtubule, and astructural change during ATP hydrolysis could shift the positions oftubulin binding sites with respect to one another, which would disruptthe microtubule lattice. Another possibility is that katanin acts morelike an ATP-regulated version of actin severing proteins, which arethought to compete for sites at protein-protein interfaces within thepolymer. In this type of mechanism, the AAA domain could serve as anATP-dependent protein clamp that binds tightly to and disruptstubulin-tubulin interfaces during particular steps in the ATPase cycle.

Targeting of Katanin to Centrosomes

Our studies show that p80 does not constitute an essential element ofkatanin's enzymatic mechanism. The finding that p80 is not required formicrotubule-severing activify was somewhat surprising, because all ofthe p60 immunoprecipitates with p80 from sea urchin cytosol (unpublishedobservations). However, experiments reported here have uncovered a rolefor p80 in targeting katanin to centrosomes in vivo. This conclusion isbased upon the finding that the WD40 domain of p80 can target GFP to thecentrosome in cultured human cell lines. Because the WD40 domain cannotdimerize with endogenous p60, the centrosomal localization must be dueto direct interaction of the WD40 domain with one or more residentcentrosomal proteins. WD40 domains are thought to form a conserved betapropeller structure, as first determined for the beta subunits oftransducin and G_(i) (Wall et al. (1995) Cell 83: 1047-1058; Sondek etal.(1996) Nature 379: 369-374). However, each WD40 domain exhibits veryspecific heterophilic protein interactions; exposed residues in the betasubunit of G_(i) interact with the alpha subunit (Wall et al. (1995)supra.), whereas the corresponding residues in the WD40 transcriptionfactor TUP1 mediate binding to a second transcription factor α2(Komachi, and Johnson (1997) Mol. Cell. Biol. 17: 6023-6028). Since theG protein beta subunits interact with multiple partner proteins (Wall etal. (1995) Cell 83: 1047-1058; Gaudet et al. (1996) Cell, 87: 577-588),it is also possible that the p80 katanin WD40 domain can interact withmore than one protein in vivo. p80 is the only known centrosomal proteinwith a WD40 motif. The findings that katanin has an entire subunitdevoted to centrosome localization and that this subunit is conservedbetween mammals and echinoderms suggest an important role for katanin atthe centrosome.

The WD40 domain of p80 katanin represents the first example of astructural motif that targets a protein to the centrosome in mammals,although a centrosome-targeting domain has been defined for theDrosophila protein CP190 (Oegema et al. (1995) J. Cell Biol. 131:1261-1273). This provides an opportunity to identify the centrosomalcomponent(s) responsible for anchoring katanin. Further information onthe docking of katanin to the centrosome may provide clues regardingkatanin's role in microtubule disassembly at this organelle.

Experimental Procedures Peptide Microsequencing

Katanin was purified from extracts of S. purpuratus eggs essentially asdescribed previous (McNally and Vale (1993) Cell, 75:419-429), exceptthat the hydroxyapatite chromatography was carried out using a PharmaciaHR10/30 column packed with 20 μm ceramic hydroxyapatite beads (AmericanInternational Chemical, Natick, Mass.). Internal peptide sequences ofthe p60 and p80 subunits were obtained from native sea urchin katanin asdescribed (Iwamatsu (1992) Electrophoresis 13: 142-147). Two additionalp80 peptides were also obtained: DASMMAM (SEQ ID NO: 14) and IQGLR (SEQID NO: 15).

p6O Cloning

A cDNA encoding a 400 bp fragment of the p60 subunit (corresponding toa.a. 214-374) was cloned from S. purpuratus first strand cDNA usingnested PCR with degenerate oligonucleotides. This fragment was then usedto screen a lambda ZAP-Express cDNA library made from S. purpuratusunfertilized egg MRNA by hybridization. Several independent positiveclones were isolated. One clone was completely sequenced (GENBANKaccession number AF052191).

p80 Cloning

An initial partial cDNA clone of p80 katanin was obtained by screeningan S. purpuratus unfertilized egg cDNA library (Wright et al. (1991) J.Cell Biol. 113: 817-833) with an antibody specific for p80 katanin,anti-p81^(aff) (McNally et al. (1996) J. Cell Sci. 109: 561-567). Theinsert of the initial clone was used to isolate a longer cDNA clone (pFM18) from the same library by plaque hybridization. A cDNA clone encodingthe 5′ end of p80 katanin (pFM23) was obtained by anchor-ligated PCR(Apte and Siebert (1993) Biotechniques 15: 890-893) using primersderived from pFM18 sequences and reverse transcription reactionsutilizing S. purpuratus unfertilized egg mRNA as template. A full-lengthp80 cDNA (GENBANK accession #AF052433) was generated by joining theinserts of pFM18 and pFM23 at a common BstX1 site.

BLAST searches of GENBANK with p80 sequences revealed homology with ahuman infant brain cDNA (GENBANK accession#: T16102) which was obtainedand sequenced. Sequences obtained from the T16102 clone were used toobtain multiple 3′ end cDNA clones by 3′ RACE from HT1080 (humanfibrosarcoma) total RNA. An overlapping cDNA clone (pFM54) containingthe translation start site was obtained by PCR amplification from anadult human hippocampal cDNA library (Stratagene, Inc.). Sequenceanalysis of partial cDNAs PCR amplified from HT1080 total RNA or fromthe hippocampal library were over 98% identical in predicted a.a.sequence. The complete DNA sequence of human p80 katanin is availablefrom GENBANK (accession #AF052432).

Antibody Production and Immunoprecipitation

The full-length S. purpuratus p60 cDNA coding sequence was inserted intopMAL-C2 (New England Biolabs) and expressed as a C-terminal fusion tomaltose binding protein in E. coli. Soluble MBP-p60 fusion protein waspurified on an amylose affinity column, eluted with maltose, andinjected into rabbits (antiserum production by BABCO, Berkely, Calif.).To select p60-specific antibodies that do not react with other AAAmembers, antibodies recognizing the N-terminal non-AAA domain of p60were affinity purified on an Affi-Gel column coupled with the N-terminalresidues 1-152 of p60 fused to MBP (Harlow and Lane (1988). Antibodies:A Laboratory Manual. (Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press)). The resulting affinity-purified antibody recognizeda single 60 kDa polypeptide in immunoblots of S. purpuratus unfertilizedegg extract.

To prepare a specific antibody to human p80 katanin, the full lengthhuman p80 cDNA was ligated into the E. coli expression vector, pET-28a⁺(Novagen) as a BamHl-XhoI fragment. The protein was expressed and thenpurified in a denatured state in 8M urea by nickel chelatechromatography on His-Bind Resin (Novagen). Rabbits were immunized withpolyacrylamide slices containing SDS-PAGE resolved human p80 katanin.Resulting serum was affinity purified with CNBr Sepharose-coupled,bacterially-expressed human p80 katanin. The resulting affinity purifiedantibody recognized a single 80 kDa polypeptide in immunoblots ofSDS-solubilized Hela cells (not shown).

For immunoprecipitations used to demonstrate association ofbaculovirus-expressed S. purpuratus p60 and p80, affinity purifiedanti-p60 antibodies were covalently crosslinked to protein A Sepharoseusing 20 mM dimethylpimilidate (Harlow and Lane (1988) supra.). Afterequilibration in TBST, 20-40:1 of antibody beads were added to kataninsamples diluted in TBST containing 1-2 mg/ml soybean trypsin inhibitor(SBTI). The immunoprecipitations were incubated at 4° C. for 1-2 hr,washed five times with 1 ml of ice-cold TBST, and eluted inSDS-containing sample buffer.

Baculovirus Expression and Purification of Katanin

Katanin subunits were expressed using the Bac-to-Bac™ baculovirusexpression system (Life Technologies), a commercial version of thesite-specific transposition system for making recombinant baculovirus(Luckow et al. (1993) J. Virology 67: 4566-4579). p60 and p80 cDNAcoding sequences were each PCR amplified (Expand polymerase, BoehringerMannheim) and then subcloned separately into pFastBac HT, which resultedin the fusion of a 6×His Ni²⁺ binding sequence to the N-terminus of bothp80 and p60. A p60-p80 coexpression virus was made by cloning thecomplete p60-FastBac HT and p86-FastBac HT coding regions into thetransfer vector, pDual. Recombinant baculovirus were prepared accordingto the Life Technologies protocol.

Sf9 cells were grown in SFM-900 II SFM (Life Technologies) supplementedwith 100× antibiotic/antimycotic (Life Technologies) to 0.5× using theshaker culture method (Weiss et al. (1995) Pp. 79-85 in BaculovirusExpression Protocols, C. D. Richardson, ed. Totowa, N.J.: Humana PressInc.). Expression of katanin subunits was performed in 11 flaskscontaining 200-300 ml of media using a multiplicity of infection of0.5-1.0 pfu/cell. The cells were harvested at approximately 72 hr postinfection by low speed centrifugation and resuspended in lysis buffer(50 mM Tris pH 8.5, 300 mM NaCl, 2 mM MgCl₂, 20 mM imidazole, 10 mM2-mercaptoethanol, 1 mM ATP, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1μg/ml aprotinin) before freezing in liquid nitrogen, and storage at −80°C.

To purify the expressed subunits, frozen cells were thawed and DNA wassheared by two passes through a Bio-Neb Cell Disrupter [100 psi helium,13 l/min]. Cell debris was removed by centrifugation (40,000 g for 45min). Subunits were bound in batch to Ni²⁺-NTA Superflow (QIAGEN),washed [20 mM Tris pH 8.0, 1 M NaCl, 2 mM MgCl₂, 40 mM imidazole, 0.02%Triton X-100, 10 mM 2-mercaptoethanol, 0.5 mM ATP] and eluted [20 mMTris pH 8.0, 100 mM NaCl, 150 mM imidazole, 2 mM MgCl₂, 0.02% TritonX-100, 10 mM 2-mercaptoethanol, 100 □M ATP], followed by freezing inliquid nitrogen. Additional purification was sometimes performed byanion-exchange chromatography. Katanin concentrations were estimated bycomparison with BSA standards using either a commercial Bradford reagent(Bio-Rad) or by densitometric analysis of Coomassie-stained SDS-PAGEgels with NIH-IMAGE after image capture on a CCD-based imaging system(Foto/Analyst, Fotodyne).

Electron Microscopic Imaging

Proteins were adsorbed to mica, freeze-dried, and platinum replicatedaccording to established procedures (Heuser (1989) J. Electron Microsc.Technique 13:, 244-263; Heuser (1983) J. Mol. Biol. 169: 155-195).Sample preparation and imaging were similar to that used in the imagingof NSF (Hanson et al. (1997) Cell 90: 523-535), except that mica flakeswere washed with a buffer consisting of 10 mM K-HEPES (pH 7.5), 2 mMMgCl₂, 1 mM nucleotide (ATP or ATP-□-S). Images were processed usingAdobe Photoshop and displayed at 300,000×.

ATPase Assays

ATPase activity was measured by a modified malachite green method(Kodama et al. (1986) J. Biochem. 99: 1465-1472). ATPase reactions of50-100 μL were carried out in a buffer previously used for measuring theATPase activity of native katanin [20 mM K-HEPES pH 8.0, 25 mMK-Glutamate, 2 mM MgCl₂, 10% glycerol (v/v), 0.02% Triton X-100 (w/v), 1mg/ml BSA] (McNally and Vale (1993) Cell, 75: 419-429), except thatsoybean trypsin inhibitor (SBTI) was replaced by BSA as a carrierprotein because SBTI increased background phosphate contamination. AnATP regenerating system consisting of 0.5-1.0 mM phospho-enol pyruvateand 2U pyruvate kinase was included to minimize the inhibition by ADPobserved previously for native katanin (McNally and Vale (1993) Cell,75; 419-429). Microtubules were prepared from bovine brain tubulin(Hyman el al. (1 990) Meth. Enzymol. 196: 303-319; Williams and Lee(1982) Meth Enzymol. 85B: 376-385). After assembly, microtubules weresedimented (230,000×g; 10 min), resuspended in ATPase buffer lackingBSA, and the polymers were resuspended by repeated passage through a 27gauge needle. Microtubule concentration was determined by measuring theabsorbance at 275 nm in 6M guanidine HCl by using a molecular mass of110 kDa and an extinction coefficient of 1.03 ml*mg⁻¹*cm⁻¹ (Hackney(1988) Proc. Natl. Acad. Sci. USA, 85: 6314-6318). ATPasc reactions werecarried out at room temperature, and were initiated by addition ofkatanin.

Severing Assays

Microscope-based severing assays were performed using previouslypublished procedures (McNally and Vale (1993) Cell, 75: 419-429), exceptthat microtubules were immobilized by first perfusing flow cells with abacterially expressed kinesin mutant that binds strongly to microtubulesbut is unable to hydrolyze ATP (K560, G234A mutant; R. Vale and E.Taylor, unpublished results). Assays were performed in 20 mM Hepes (pH7.5), 2 mM MgCl₂, 1 mM ATP with an oxygen scavenger system consisting ofglucose oxidase (220 μg/ml), catalase (36 μg/ml), glucose (22.5 mM), and2-mercaptoethanol (71.5 mM). Images were captured using a cooled,slow-scan CCD (Photometerics) and processed using Adobe Photoshop.

DAPI severing assays were performed using conditions where the change influorescence intensity was linear with the amount of tubulin polymeradded (Heusele et al. (1987) Eur. J. Biochem. 165: 613-620). Severingreactions contained 2 μM microtubules (polymerized and resuspended inATPase buffer as above) were incubated with 10 μM DAPI, along with 1 mMATP, 10 mM phospho-enol pyruvate, 250 μg/ml pyruvate kinase (BoehringerMannheim), and 1 mg/ml BSA. The reaction volume was 80 μL, and.fluorescence intensity was measured by exciting at 370 nm and measuringthe emission at 450 nm using a model 8100 fluorimeter. (SLM Instruments)in photon counting mode.

In vitro Translation Co-Immunoprecipitation

In order to facilitate the non-radioactive detection of in vitrotranslated p60 and p80, each cDNA was ligated into the vector pCITE-4a+(Novagen) such that the proteins would be translated in frame with a 37amino acid N-terminal S-Tag. In vitro synthesis of proteins directlyfrom plasmid DNAs was accomplished using the Single Tube Protein System2, T7 (Novagen). For co-immunoprecipitation assays, p60 and p80constructs were usually co-expressed. However, identical results wereobtained if the constructs were expressed separately and then incubatedtogether for 30 min at room temperature. For immunoprecipitations,lysates were incubated on ice with Pansorbin (Calbiochem)-antibodycomplexes, washed in NET buffer (50 mM Tris-Cl (pH 7.5), 150 mM NaCl,0.1% Nonidet P-40, 1 mM EDTA (pH 8.0), 0.25% gelatin 0.02% sodiumazide), then resuspended in SDS-PAGE sample buffer. In vitro translationproducts in both the pellets and supernatants from theimmunoprecipitations were resolved by SDS-PAGE, transferred tonitrocellulose, probed with S-protein HRP conjugate (Novagen) anddetected by chemiluminescence.

Cell Culture and Transfections and Immunofluorescence

To allow transient expression of a human p80 WD40-GFP fusion protein inHeLa cells, a DNA fragment containing amino acids 1-263 of human p80katanin was generated by PCR amplification, placing a BamHl site and aKozak consensus at the predicted translation start and an EcoRI siteafter the codon for a.a. 263. This BamH1-EcoR1 fragment was ligated intothe GFP fusion vector pEGFP-N1 (Clontech).

Both MSU1.1 and Hela cells were grown on 18 mm glass coverslips inOptimem medium (Life Technologies) supplemented with 10% fetal bovineserum, penicillin and streptomycin. Plasmids were transfected usingSuperfect Reagent (Qiagen) for 2 hr after which coverslips were washedwith PBS and placed in fresh culture medium at 37° C. with 5% CO₂ for1-24 hr.

For imaging of GFP-fluorescence and immunofluorescence with the humanp80 katanin antibody or with the γ-tubulin antibody, monoclonal GTU88(Sigma Chemical), cells on coverslips were fixed either in −20° C.methanol or in 0.5×PBS, 3.7% formaldehyde, 75% methanol at 22° C. for 10min followed by rehydration in TBST. Antibody labelling was carried outin TBST containing 4% BSA. Images were captured with a Nikon MicrophotSA microscope, 100× Plan Fluor 1.3 objective, Photometrics Quantixcamera and IP Lab Spectrum software (Scanalytics).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

15 1 517 PRT Strongylocentrotus purpuratus misc_feature katanin p60subunit 1 Met Ser Val Asp Glu Ile Cys Glu Asn Thr Lys Met Gly Arg GluTyr 1 5 10 15 Ala Leu Leu Gly Asn Tyr Glu Thr Ser Leu Val Tyr Tyr GlnGly Val 20 25 30 Leu Gln Gln Ile Gln Lys Leu Leu Thr Ser Val His Glu ProGln Arg 35 40 45 Lys His Gln Trp Gln Thr Ile Arg Gln Glu Leu Ser Gln GluTyr Glu 50 55 60 His Val Lys Asn Ile Thr Lys Thr Leu Asn Gly Phe Lys SerGlu Pro 65 70 75 80 Ala Ala Pro Glu Pro Ala Pro Asn His Gly Arg Ala AlaPro Phe Ser 85 90 95 His His Gln His Ala Ala Lys Pro Ala Ala Ala Glu ProAla Arg Asp 100 105 110 Pro Asp Val Trp Pro Pro Pro Thr Pro Val Asp HisArg Pro Ser Pro 115 120 125 Pro Tyr Gln Arg Ala Ala Arg Lys Asp Pro ProArg Arg Ser Glu Pro 130 135 140 Ser Lys Pro Ala Asn Arg Ala Pro Gly AsnAsp Arg Gly Gly Arg Gly 145 150 155 160 Pro Ser Asp Arg Arg Gly Asp AlaArg Ser Gly Gly Gly Gly Arg Gly 165 170 175 Gly Ala Arg Gly Ser Asp LysAsp Lys Asn Arg Gly Gly Lys Ser Asp 180 185 190 Lys Asp Lys Lys Ala ProSer Gly Glu Glu Gly Asp Glu Lys Lys Phe 195 200 205 Asp Pro Ala Gly TyrAsp Lys Asp Leu Val Glu Asn Leu Glu Arg Asp 210 215 220 Ile Val Gln ArgAsn Pro Asn Val His Trp Ala Asp Ile Ala Gly Leu 225 230 235 240 Thr GluAla Lys Arg Leu Leu Glu Glu Ala Val Val Leu Pro Leu Trp 245 250 255 MetPro Asp Tyr Phe Lys Gly Ile Arg Arg Pro Trp Lys Gly Val Leu 260 265 270Met Val Gly Pro Pro Gly Thr Gly Lys Thr Met Leu Ala Lys Ala Val 275 280285 Ala Thr Glu Cys Gly Thr Thr Phe Phe Asn Val Ser Ser Ala Ser Leu 290295 300 Thr Ser Lys Tyr His Gly Glu Ser Glu Lys Leu Val Arg Leu Leu Phe305 310 315 320 Glu Met Ala Arg Phe Tyr Ala Pro Ser Thr Ile Phe Ile AspGlu Ile 325 330 335 Asp Ser Ile Cys Ser Lys Arg Gly Thr Gly Ser Glu HisGlu Ala Ser 340 345 350 Arg Arg Val Lys Ser Glu Leu Leu Ile Gln Met AspGly Val Ser Gly 355 360 365 Pro Ser Ala Gly Glu Glu Ser Ser Lys Met ValMet Val Leu Ala Ala 370 375 380 Thr Asn Phe Pro Trp Asp Ile Asp Glu AlaLeu Arg Arg Arg Leu Glu 385 390 395 400 Lys Arg Ile Tyr Ile Pro Leu ProGlu Ile Asp Gly Arg Glu Gln Leu 405 410 415 Leu Arg Ile Asn Leu Lys GluVal Pro Leu Ala Asp Asp Ile Asp Leu 420 425 430 Lys Ser Ile Ala Glu LysMet Asp Gly Tyr Ser Gly Ala Asp Ile Thr 435 440 445 Asn Val Cys Arg AspAla Ser Met Met Ala Met Arg Arg Arg Ile Gln 450 455 460 Gly Leu Arg ProGlu Glu Ile Arg His Ile Pro Lys Glu Glu Leu Asn 465 470 475 480 Gln ProSer Thr Pro Ala Asp Phe Leu Leu Ala Leu Gln Lys Val Ser 485 490 495 LysSer Val Gly Lys Glu Asp Leu Val Lys Tyr Met Ala Trp Met Glu 500 505 510Glu Phe Gly Ser Val 515 2 690 PRT Strongylocentrotus purpuratusmisc_feature katanin p80 subunit 2 Met Ala Thr Lys Arg Ala Trp Lys LeuGln Glu Leu Val Ala His Ser 1 5 10 15 Ser Asn Val Asn Cys Leu Ala LeuGly Pro Met Ser Gly Arg Val Met 20 25 30 Val Thr Gly Gly Glu Asp Lys LysVal Asn Leu Trp Ala Val Gly Lys 35 40 45 Gln Asn Cys Ile Ile Ser Leu SerGly His Thr Ser Pro Val Asp Ser 50 55 60 Val Lys Phe Asn Ser Ser Glu GluLeu Val Val Ala Gly Ser Gln Ser 65 70 75 80 Gly Thr Met Lys Ile Tyr AspLeu Glu Pro Ala Lys Ile Val Arg Thr 85 90 95 Leu Thr Gly His Arg Asn SerIle Arg Cys Met Asp Phe His Pro Phe 100 105 110 Gly Glu Phe Val Ala SerGly Ser Thr Asp Thr Asn Val Lys Leu Trp 115 120 125 Asp Val Arg Arg LysGly Cys Ile Tyr Thr Tyr Lys Gly His Ser Asp 130 135 140 Gln Val Asn MetIle Lys Phe Ser Pro Asp Gly Lys Trp Leu Val Thr 145 150 155 160 Ala SerGlu Asp Thr Thr Ile Lys Leu Trp Asp Leu Thr Met Gly Lys 165 170 175 LeuPhe Gln Glu Phe Lys Asn His Thr Gly Gly Val Thr Gly Ile Glu 180 185 190Phe His Pro Asn Glu Phe Leu Leu Ala Ser Gly Ser Ser Asp Arg Thr 195 200205 Val Gln Phe Trp Asp Leu Glu Thr Phe Gln Leu Val Ser Ser Thr Ser 210215 220 Pro Gly Ala Ser Ala Val Arg Ser Ile Ser Phe His Pro Asp Gly Ser225 230 235 240 Tyr Leu Phe Cys Ser Ser Gln Asp Met Leu His Ala Phe GlyTrp Glu 245 250 255 Pro Ile Arg Cys Phe Asp Thr Phe Ser Val Phe Trp GlyLys Val Ala 260 265 270 Asp Thr Val Ile Ala Ser Thr Gln Leu Ile Gly AlaSer Phe Asn Ala 275 280 285 Thr Asn Val Ser Val Tyr Val Ala Asp Leu SerArg Met Ser Thr Thr 290 295 300 Gly Ile Ala Gln Glu Pro Gln Ser Gln ProSer Lys Thr Pro Ser Gly 305 310 315 320 Gly Ala Glu Glu Val Pro Ser LysPro Leu Thr Ala Ser Gly Arg Lys 325 330 335 Asn Phe Val Arg Glu Arg ProHis Thr Thr Ser Ser Lys Gln Arg Gln 340 345 350 Pro Asp Val Lys Ser GluPro Glu Arg Gln Ser Pro Thr Gln Asp Glu 355 360 365 Gly Val Lys Asp AspAsp Ala Thr Asp Ile Lys Asp Pro Asp Ser Tyr 370 375 380 Ala Lys Ile PheSer Pro Lys Thr Arg Val Asp His Ser Pro Glu Arg 385 390 395 400 Asn AlaGln Pro Phe Pro Ala Pro Leu Asp Val Pro Gly Ala Gln Glu 405 410 415 ProGlu Pro Phe Lys His Pro Pro Lys Pro Ala Ala Ala Ala Ala Val 420 425 430Ala Pro Val Ser Arg Ala Pro Ala Pro Ser Ala Ser Asp Trp Gln Pro 435 440445 Ala Gln Ala Asn Pro Ala Pro Asn Arg Val Pro Ala Ala Thr Lys Pro 450455 460 Val Pro Ala Gln Glu Val Ala Pro Ser Arg Lys Pro Asp Pro Ile Ser465 470 475 480 Thr Ile Ile Pro Ser Asp Arg Asn Lys Pro Ala Asn Leu AspMet Asp 485 490 495 Ala Phe Leu Pro Pro Ala His Ala Gln Gln Ala Pro ArgVal Asn Ala 500 505 510 Pro Ala Ser Arg Lys Gln Ser Asp Ser Glu Arg IleGlu Gly Leu Arg 515 520 525 Lys Gly His Asp Ser Met Cys Gln Val Leu SerSer Arg His Arg Asn 530 535 540 Leu Asp Val Val Arg Ala Ile Trp Thr AlaGly Asp Ala Lys Thr Ser 545 550 555 560 Val Glu Ser Val Val Asn Met LysAsp Gln Ala Ile Leu Val Asp Ile 565 570 575 Leu Asn Ile Met Leu Leu LysLys Ser Leu Trp Asn Leu Asp Met Cys 580 585 590 Val Val Val Leu Pro ArgLeu Lys Glu Leu Leu Ser Ser Lys Tyr Glu 595 600 605 Asn Tyr Val His ThrSer Cys Ala Cys Leu Lys Leu Ile Leu Lys Asn 610 615 620 Phe Thr Ser LeuPhe Asn Gln Asn Ile Lys Cys Pro Pro Ser Gly Ile 625 630 635 640 Asp IleThr Arg Glu Glu Arg Tyr Asn Lys Cys Ser Lys Cys Tyr Ser 645 650 655 TyrLeu Ile Ala Thr Arg Gly Tyr Val Glu Glu Lys Gln His Val Ser 660 665 670Gly Lys Leu Gly Ser Ser Phe Arg Glu Leu His Leu Leu Leu Asp Gln 675 680685 Leu Glu 690 3 730 PRT Xenopus laevis misc_feature Xenopus kinesincentral motor 1 (XKCM1) 3 Met Glu Arg Leu Val Ala Thr Arg Leu Val ThrGly Leu Ala Val Lys 1 5 10 15 Ile Met Arg Ser Asn Gly Val Ile His AsnAla Asn Ile Thr Ser Val 20 25 30 Asn Met Asp Arg Ser Ser Val Asn Val GluTrp Lys Glu Gly Glu Ala 35 40 45 Asn Lys Gly Lys Glu Ile Ser Phe Ala AspVal Ile Ser Val Asn Pro 50 55 60 Glu Leu Leu Asp Ala Val Leu Ala Pro ThrAsn Val Lys Glu Asn Met 65 70 75 80 Pro Pro Gln Arg Asn Val Ser Ser GlnAsn His Lys Arg Lys Thr Ile 85 90 95 Ser Lys Ile Pro Ala Pro Lys Glu ValAla Ala Lys Asn Ser Leu Leu 100 105 110 Ser Glu Ser Gly Ala Gln Ser ValLeu Arg Glu Arg Ser Thr Arg Met 115 120 125 Thr Ala Ile His Glu Thr LeuPro Tyr Glu Asn Glu Met Glu Ala Glu 130 135 140 Ser Thr Pro Leu Pro IleGln Gln Asn Ser Val Gln Ala Arg Ser Arg 145 150 155 160 Ser Thr Lys ValSer Ile Ala Glu Glu Pro Arg Leu Gln Thr Arg Ile 165 170 175 Ser Glu IleVal Glu Glu Ser Leu Pro Ser Gly Arg Asn Asn Gln Gly 180 185 190 Arg ArgLys Ser Asn Ile Val Lys Glu Met Glu Lys Met Lys Asn Lys 195 200 205 ArgGlu Glu Gln Arg Ala Gln Asn Tyr Glu Arg Arg Met Lys Arg Ala 210 215 220Gln Asp Tyr Asp Thr Ser Val Pro Asn Trp Glu Phe Gly Lys Met Ile 225 230235 240 Lys Glu Phe Arg Ala Thr Met Asp Cys His Arg Ile Ser Met Ala Asp245 250 255 Pro Ala Glu Glu His Arg Ile Cys Val Cys Val Arg Lys Arg ProLeu 260 265 270 Asn Lys Gln Glu Leu Ser Lys Lys Glu Ile Asp Ile Ile SerVal Pro 275 280 285 Ser Lys Asn Ile Val Leu Val His Glu Pro Lys Leu LysVal Asp Leu 290 295 300 Thr Lys Tyr Leu Glu Asn Gln Ala Phe Arg Phe AspPhe Ser Phe Asp 305 310 315 320 Glu Thr Ala Thr Asn Glu Val Val Tyr ArgPhe Thr Ala Arg Pro Leu 325 330 335 Val Gln Ser Ile Phe Glu Gly Gly LysAla Thr Cys Phe Ala Tyr Gly 340 345 350 Gln Thr Gly Ser Gly Lys Thr HisThr Met Gly Gly Asp Phe Ser Gly 355 360 365 Lys Ser Gln Asn Val Ser LysGly Val Tyr Ala Phe Ala Ser Arg Asp 370 375 380 Val Phe Leu Leu Leu AspGln Pro Arg Tyr Lys His Leu Asp Leu Asp 385 390 395 400 Val Phe Val ThrPhe Phe Glu Ile Tyr Asn Gly Lys Val Phe Asp Leu 405 410 415 Leu Asn LysLys Thr Lys Leu Arg Val Leu Glu Asp Ala Lys Gln Glu 420 425 430 Val GlnVal Val Gly Leu Leu Glu Lys Gln Val Ile Ser Ala Asp Asp 435 440 445 ValPhe Lys Met Ile Glu Ile Gly Ser Ala Cys Arg Thr Ser Gly Gln 450 455 460Thr Phe Ala Asn Thr Ser Ser Ser Arg Ser His Ala Cys Leu Gln Ile 465 470475 480 Ile Leu Arg Arg Gly Ser Lys Leu His Gly Lys Phe Ser Leu Val Asp485 490 495 Leu Ala Gly Asn Glu Arg Gly Val Asp Thr Ala Ser Ala Asp ArgIle 500 505 510 Thr Arg Met Lys Gly Ala Glu Ile Asn Arg Ser Leu Leu AlaLeu Lys 515 520 525 Glu Cys Ile Arg Ala Leu Gly Gln Asn Lys Ser His ThrPro Phe Arg 530 535 540 Glu Ser Lys Leu Thr Gln Ile Leu Arg Asp Ser PheIle Gly Glu Asn 545 550 555 560 Ser Arg Thr Cys Met Ile Ala Met Leu SerPro Gly Phe Asn Ser Cys 565 570 575 Glu Tyr Thr Leu Asn Thr Leu Arg TyrAla Asp Arg Val Lys Glu Leu 580 585 590 Ser Pro Gln Asn Ala Glu Thr AsnAsp Asp Asn Leu Gln Met Glu Asp 595 600 605 Ser Gly Gly Ser His Ala SerIle Glu Gly Leu Gln Leu Gln Asp Asp 610 615 620 Phe Leu Leu Lys Asp GluGlu Leu Ser Thr His Asn Ser Phe Gln Asp 625 630 635 640 Ala Leu Asn ArgVal Gly Glu Leu Glu Asp Lys Ala Val Asp Glu Leu 645 650 655 Arg Glu LeuVal Gln Lys Glu Pro Glu Trp Thr Asn Leu Leu Gln Met 660 665 670 Thr GluGln Pro Asp Tyr Asp Leu Glu Asn Phe Val Met Gln Ala Glu 675 680 685 TyrLeu Ile Gln Glu Arg Ser Lys Val Leu Ile Ala Leu Gly Asp Ser 690 695 700Ile Asn Ser Leu Arg Leu Ala Leu Gln Val Glu Glu Gln Ala Ser Lys 705 710715 720 Gln Ile Ser Lys Lys Lys Arg Ser Asn Lys 725 730 4 217 PRTStrongylocentrotus purpuratus misc_feature AAA ATPase superfamilykatanin p60 AAA domain 4 Val His Trp Ala Asp Ile Ala Gly Leu Thr Glu AlaLys Arg Leu Leu 1 5 10 15 Glu Glu Ala Val Val Leu Pro Leu Trp Met ProAsp Tyr Phe Lys Gly 20 25 30 Ile Phe Phe Pro Trp Lys Gly Val Leu Met ValGly Pro Pro Gly Thr 35 40 45 Gly Lys Thr Met Leu Ala Lys Ala Val Ala ThrGlu Cys Gly Thr Thr 50 55 60 Phe Phe Asn Val Ser Ser Ala Ser Leu Thr SerLys Tyr His Gly Glu 65 70 75 80 Ser Glu Lys Leu Val Arg Leu Leu Phe GluMet Ala Arg Phe Tyr Ala 85 90 95 Pro Ser Thr Ile Phe Ile Asp Glu Ile AspSer Ile Cys Ser Lys Arg 100 105 110 Gly Thr Gly Ser Glu His Glu Ala SerArg Arg Val Lys Ser Glu Leu 115 120 125 Leu Ile Gln Met Asp Gly Val SerGly Pro Ser Ala Gly Glu Glu Ser 130 135 140 Ser Lys Met Val Met Val LeuAla Ala Thr Asn Phe Pro Trp Asp Ile 145 150 155 160 Asp Glu Ala Leu ArgArg Arg Leu Glu Lys Arg Ile Tyr Ile Pro Leu 165 170 175 Pro Glu Ile AspGly Arg Glu Gln Leu Leu Arg Ile Asn Leu Lys Glu 180 185 190 Val Pro LeuAla Asp Asp Ile Asp Leu Lys Ser Ile Ala Glu Lys Met 195 200 205 Asp GlyTyr Ser Gly Ala Asp Ile Thr 210 215 5 213 PRT Caenorhabditis elegansmisc_feature AAA ATPase superfamily mei-1 AAA domain 5 Met Ser Leu AspAsp Ile Ile Gly Met His Asp Val Lys Gln Val Leu 1 5 10 15 His Glu AlaVal Thr Leu Pro Leu Leu Val Pro Glu Phe Phe Gln Gly 20 25 30 Leu Arg SerPro Trp Lys Ala Met Val Leu Ala Gly Pro Pro Gly Thr 35 40 45 Gly Lys ThrLeu Ile Ala Arg Ala Ile Ala Ser Glu Ser Ser Ser Thr 50 55 60 Phe Phe ThrVal Ser Ser Thr Asp Leu Ser Ser Lys Trp Arg Gly Asp 65 70 75 80 Ser GluLys Ile Val Arg Leu Leu Phe Glu Leu Ala Arg Phe Tyr Ala 85 90 95 Pro SerIle Ile Phe Ile Asp Glu Ile Asp Thr Leu Gly Gly Gln Arg 100 105 110 GlyAsn Ser Gly Glu His Glu Ala Ser Arg Arg Val Lys Ser Glu Phe 115 120 125Leu Val Gln Met Asp Gly Ser Gln Asn Lys Phe Asp Ser Arg Arg Val 130 135140 Phe Val Leu Ala Ala Thr Asn Ile Pro Trp Glu Leu Asp Glu Ala Leu 145150 155 160 Arg Arg Arg Phe Glu Lys Arg Ile Phe Ile Pro Leu Pro Asp IleAsp 165 170 175 Ala Arg Lys Lys Leu Ile Glu Lys Ser Met Glu Gly Thr ProLys Ser 180 185 190 Asp Glu Ile Asn Tyr Asp Asp Leu Ala Ala Arg Thr GluGly Phe Ser 195 200 205 Gly Ala Asp Val Val 210 6 215 PRT Saccharomycescerevisiae misc_feature AAA ATPase superfamily sug1 AAA domain 6 Ser ThrTyr Asp Met Val Gly Gly Leu Thr Lys Gln Ile Lys Glu Ile 1 5 10 15 LysGlu Val Ile Glu Leu Pro Val Lys His Pro Glu Leu Phe Glu Ser 20 25 30 LeuGly Ile Ala Gln Pro Lys Gly Val Ile Leu Tyr Gly Pro Pro Gly 35 40 45 ThrGly Lys Thr Leu Leu Ala Arg Ala Val Ala His His Thr Asp Cys 50 55 60 LysPhe Ile Arg Val Ser Gly Ala Glu Leu Val Gln Lys Tyr Ile Gly 65 70 75 80Glu Gly Ser Arg Met Val Arg Glu Leu Phe Val Met Ala Arg Glu His 85 90 95Ala Pro Ser Ile Ile Phe Met Asp Glu Ile Asp Ser Ile Gly Ser Thr 100 105110 Arg Val Glu Gly Ser Gly Gly Gly Asp Ser Glu Val Gln Arg Thr Met 115120 125 Leu Glu Leu Leu Asn Gln Leu Asp Gly Phe Glu Thr Ser Lys Asn Ile130 135 140 Lys Ile Ile Met Ala Thr Asn Arg Leu Asp Ile Leu Asp Pro AlaLeu 145 150 155 160 Leu Arg Pro Gly Arg Ile Asp Arg Lys Ile Glu Phe ProPro Pro Ser 165 170 175 Val Ala Ala Arg Ala Glu Ile Leu Arg Ile His SerArg Lys Met Asn 180 185 190 Leu Thr Arg Gly Ile Asn Leu Arg Lys Val AlaGlu Lys Met Asn Gly 195 200 205 Cys Ser Gly Ala Asp Val Lys 210 215 7214 PRT Escherichia coli misc_feature AAA ATPase superfamily ftsH AAAdomain 7 Thr Thr Phe Ala Asp Val Ala Gly Cys Asp Glu Ala Lys Glu Glu Val1 5 10 15 Ala Glu Leu Val Glu Tyr Leu Arg Glu Pro Ser Arg Phe Gln LysLeu 20 25 30 Gly Gly Lys Glu Pro Lys Gly Val Leu Met Val Gly Pro Pro GlyThr 35 40 45 Gly Lys Thr Leu Leu Ala Lys Ala Ile Ala Gly Glu Ala Lys ValPro 50 55 60 Phe Phe Thr Ile Ser Gly Ser Asp Phe Val Glu Met Phe Val GlyVal 65 70 75 80 Gly Ala Ser Arg Val Arg Asp Met Phe Glu Gln Ala Lys LysAla Ala 85 90 95 Pro Cys Ile Ile Phe Ile Asp Glu Ile Asp Ala Val Gly ArgGln Arg 100 105 110 Gly Ala Gly Leu Gly Gly Gly His Asp Glu Arg Glu GlnThr Leu Asn 115 120 125 Gln Met Leu Val Glu Met Asp Gly Phe Glu Gly AsnGlu Gly Ile Ile 130 135 140 Val Ile Ala Ala Thr Asn Arg Pro Asp Val LeuAsp Pro Ala Leu Leu 145 150 155 160 Arg Pro Gly Arg Phe Asp Arg Gln ValVal Val Gly Leu Pro Asp Val 165 170 175 Arg Gly Arg Glu Gln Ile Leu LysVal His Met Arg Arg Val Pro Leu 180 185 190 Ala Pro Asp Ile Asp Ala AlaIle Ile Ala Arg Gly Thr Pro Gly Phe 195 200 205 Ser Gly Ala Asp Leu Ala210 8 221 PRT Saccharomyces cerevisiae misc_feature AAA ATPasesuperfamily PAS1 AAA domain 8 Ile Lys Trp Gly Asp Ile Gly Ala Leu AlaAsn Ala Lys Asp Val Leu 1 5 10 15 Leu Glu Thr Leu Glu Trp Pro Thr LysTyr Glu Pro Ile Phe Val Asn 20 25 30 Cys Pro Leu Arg Leu Arg Ser Gly IleLeu Leu Tyr Gly Tyr Pro Gly 35 40 45 Cys Gly Lys Thr Leu Leu Ala Ser AlaVal Ala Gln Gln Cys Gly Leu 50 55 60 Asn Phe Ile Ser Val Lys Gly Pro GluIle Leu Asn Lys Phe Ile Gly 65 70 75 80 Ala Ser Glu Gln Asn Ile Arg GluLeu Phe Glu Arg Ala Gln Ser Val 85 90 95 Lys Pro Cys Ile Leu Phe Phe AspGlu Phe Asp Ser Ile Ala Pro Lys 100 105 110 Arg Gly His Asp Ser Thr GlyVal Thr Asp Arg Val Val Asn Gln Leu 115 120 125 Leu Thr Gln Met Asp GlyAla Glu Gly Leu Asp Gly Val Tyr Ile Leu 130 135 140 Ala Ala Thr Ser ArgPro Asp Leu Ile Asp Ser Ala Leu Leu Arg Pro 145 150 155 160 Gly Arg LeuAsp Lys Ser Val Ile Cys Asn Ile Pro Thr Glu Ser Glu 165 170 175 Arg LeuAsp Ile Leu Gln Ala Ile Val Asn Ser Lys Asp Lys Asp Thr 180 185 190 GlyGln Lys Lys Phe Ala Leu Glu Lys Asn Ala Asp Leu Lys Leu Ile 195 200 205Ala Glu Lys Thr Ala Gly Phe Ser Gly Ala Asp Leu Gln 210 215 220 9 227PRT Cricetulus longicaudatus misc_feature AAA ATPase superfamilyN-ethylmaleimide sensitive fusion protein (NSF) AAA domai 9 Glu Lys MetGly Ile Gly Gly Leu Asp Lys Glu Phe Ser Asp Ile Phe 1 5 10 15 Arg ArgAla Phe Ala Ser Arg Val Phe Pro Pro Glu Ile Val Glu Gln 20 25 30 Met GlyCys Lys His Val Lys Gly Ile Leu Leu Tyr Gly Pro Pro Gly 35 40 45 Cys GlyLys Thr Leu Leu Ala Arg Gln Ile Gly Lys Met Leu Asn Ala 50 55 60 Arg GluPro Lys Val Val Asn Gly Pro Glu Ile Leu Asn Lys Tyr Val 65 70 75 80 GlyGlu Ser Glu Ala Asn Ile Arg Lys Leu Phe Ala Asp Ala Glu Glu 85 90 95 GluGln Arg Arg Leu Gly Ala Asn Ser Gly Leu His Ile Ile Ile Phe 100 105 110Asp Glu Ile Asp Ala Ile Cys Lys Gln Arg Gly Ser Met Ala Gly Ser 115 120125 Thr Gly Val His Asp Thr Val Val Asn Gln Leu Leu Ser Lys Ile Asp 130135 140 Gly Val Glu Gln Leu Asn Asn Ile Leu Val Ile Gly Met Thr Asn Arg145 150 155 160 Pro Asp Leu Ile Asp Glu Ala Leu Leu Arg Pro Gly Arg LeuGlu Val 165 170 175 Lys Met Glu Ile Gly Leu Pro Asp Glu Lys Gly Arg LeuGln Ile Leu 180 185 190 His Ile His Thr Ala Arg Met Arg Gly His Gln LeuLeu Ser Ala Asp 195 200 205 Val Asp Ile Lys Glu Leu Ala Val Glu Thr LysAsn Phe Ser Gly Ala 210 215 220 Glu Leu Glu 225 10 253 PRTStrongylocentrotus purpuratus misc_feature katanin p80 subunit WD40repeat region 10 Lys Arg Ala Trp Lys Leu Gln Glu Leu Val Ala His Ser SerAsn Val 1 5 10 15 Asn Cys Leu Ala Leu Gly Pro Met Ser Gly Arg Val MetVal Thr Gly 20 25 30 Gly Glu Asp Lys Lys Val Asn Leu Trp Ala Val Gly LysGln Asn Cys 35 40 45 Ile Ile Ser Leu Ser Gly His Thr Ser Pro Val Asp SerVal Lys Phe 50 55 60 Asn Ser Ser Glu Glu Leu Val Val Ala Gly Ser Gln SerGly Thr Met 65 70 75 80 Lys Ile Tyr Asp Leu Glu Pro Ala Lys Ile Val ArgThr Leu Thr Gly 85 90 95 His Arg Asn Ser Ile Arg Cys Met Asp Phe His ProPhe Gly Glu Phe 100 105 110 Val Ala Ser Gly Ser Thr Asp Thr Asn Val LysLeu Trp Asp Val Arg 115 120 125 Arg Lys Gly Cys Ile Tyr Thr Tyr Lys GlyHis Ser Asp Gln Val Asn 130 135 140 Met Ile Lys Phe Ser Pro Asp Gly LysTrp Leu Val Thr Ala Ser Glu 145 150 155 160 Asp Thr Thr Ile Lys Glu TrpAsp Leu Thr Met Gly Lys Leu Phe Gln 165 170 175 Glu Phe Lys Asn His ThrGly Gly Val Thr Gly Ile Glu Phe His Pro 180 185 190 Asn Glu Phe Leu LeuAla Ser Gly Ser Ser Asp Arg Thr Val Gln Phe 195 200 205 Trp Asp Leu GluThr Phe Gln Leu Val Ser Ser Thr Ser Pro Gly Ala 210 215 220 Ser Ala ValArg Ser Ile Ser Phe His Pro Asp Gly Ser Tyr Leu Phe 225 230 235 240 CysSer Ser Gln Asp Met Leu His Ala Phe Gly Trp Glu 245 250 11 253 PRT Homosapiens misc_feature putative human ortholog of katanin p80 (Hs p80)WD40 repeat regio 11 Lys Thr Ala Trp Lys Leu Gln Glu Ile Val Ala His AlaSer Asn Val 1 5 10 15 Ser Ser Leu Val Leu Gly Lys Ala Ser Gly Arg LeuLeu Ala Thr Gly 20 25 30 Gly Asp Asp Cys Arg Val Asn Leu Trp Ser Ile AsnLys Pro Asn Cys 35 40 45 Ile Met Ser Leu Thr Gly His Thr Ser Pro Val GluSer Val Arg Leu 50 55 60 Asn Thr Pro Glu Glu Leu Ile Val Ala Gly Ser GlnSer Gly Ser Ile 65 70 75 80 Arg Val Trp Asp Leu Glu Ala Ala Lys Ile LeuArg Thr Leu Met Gly 85 90 95 Leu Lys Ala Asn Ile Cys Ser Leu Asp Phe HisPro Tyr Gly Glu Phe 100 105 110 Val Ala Ser Gly Ser Gln Asp Thr Asn IleLys Leu Trp Asp Ile Arg 115 120 125 Arg Lys Gly Cys Val Phe Arg Tyr ArgGly His Ser Gln Ala Val Arg 130 135 140 Cys Leu Arg Phe Ser Pro Asp GlyLys Trp Leu Ala Ser Ala Ala Asp 145 150 155 160 Asp His Thr Val Glu LeuTrp Asp Leu Thr Ala Gly Lys Met Met Ser 165 170 175 Glu Phe Pro Gly HisThr Gly Pro Val Asn Val Val Glu Phe His Pro 180 185 190 Asn Glu Tyr LeuLeu Ala Ser Gly Ser Ser Asp Gly Thr Ile Arg Phe 195 200 205 Trp Asp LeuGlu Lys Phe Gln Val Val Ser Arg Ile Glu Gly Glu Pro 210 215 220 Gly ProVal Arg Ser Val Leu Phe Asn Pro Asp Gly Cys Cys Leu Tyr 225 230 235 240Ser Gly Cys Gln Asp Ser Leu Arg Val Tyr Gly Trp Glu 245 250 12 250 PRTHomo sapiens misc_feature TFIID WD40 repeat region 12 Lys Thr Ala SerGlu Leu Lys Ile Leu Tyr Gly His Ser Gly Pro Val 1 5 10 15 Tyr Gly AlaSer Phe Ser Pro Asp Arg Asn Tyr Leu Leu Ser Ser Ser 20 25 30 Glu Asp GlyThr Val Arg Leu Trp Ser Leu Gln Thr Phe Thr Cys Leu 35 40 45 Val Gly TyrLys Gly His Asn Tyr Pro Val Trp Asp Thr Gln Phe Ser 50 55 60 Pro Tyr GlyTyr Tyr Phe Val Ser Gly Gly His Asp Arg Val Ala Arg 65 70 75 80 Leu TrpAla Thr Asp His Tyr Gln Pro Leu Arg Ile Phe Ala Gly His 85 90 95 Leu AlaAsp Val Asn Cys Thr Arg Phe His Pro Asn Ser Asn Tyr Val 100 105 110 AlaThr Gly Ser Ala Asp Arg Thr Val Arg Leu Trp Asp Val Leu Asn 115 120 125Gly Asn Cys Val Arg Ile Phe Thr Gly His Lys Gly Pro Ile His Ser 130 135140 Leu Thr Phe Ser Pro Asn Gly Arg Phe Leu Ala Thr Gly Ala Thr Asp 145150 155 160 Gly Arg Val Leu Leu Trp Asp Ile Gly His Gly Leu Met Val GlyGlu 165 170 175 Leu Lys Gly His Thr Asp Thr Val Cys Ser Leu Arg Phe SerArg Asp 180 185 190 Gly Glu Ile Leu Ala Ser Gly Ser Met Asp Asn Thr ValArg Leu Trp 195 200 205 Asp Ala Ile Lys Ala Phe Glu Asp Leu Glu Thr AspAsp Phe Thr Thr 210 215 220 Ala Thr Gly His Ile Asn Leu Pro Glu Asn SerGln Glu Leu Leu Leu 225 230 235 240 Gly Thr Tyr Met Thr Lys Ser Thr ProVal 245 250 13 251 PRT Thermomonospora curvata misc_feature putativeserine/threonine kinase PkwA WD40 repeat region 13 Ala Ser Gly Asp GluLeu His Thr Leu Glu Gly His Thr Asp Trp Val 1 5 10 15 Arg Ala Val AlaPhe Ser Pro Asp Gly Ala Leu Leu Ala Ser Gly Ser 20 25 30 Asp Asp Ala ThrVal Arg Leu Trp Asp Val Ala Ala Ala Glu Glu Arg 35 40 45 Ala Val Phe GluGly His Thr His Tyr Val Leu Asp Ile Ala Phe Ser 50 55 60 Pro Asp Gly SerMet Val Ala Ser Gly Ser Arg Asp Gly Thr Ala Arg 65 70 75 80 Leu Trp AsnVal Ala Thr Gly Thr Glu His Ala Val Leu Lys Gly His 85 90 95 Thr Asp TyrVal Tyr Ala Val Ala Phe Ser Pro Asp Gly Ser Met Val 100 105 110 Ala SerGly Ser Arg Asp Gly Thr Ile Arg Leu Trp Asp Val Ala Thr 115 120 125 GlyLys Glu Arg Asp Val Leu Gln Ala Pro Ala Glu Asn Val Val Ser 130 135 140Leu Ala Phe Ser Pro Asp Gly Ser Met Leu Val His Gly Ser Asp Ser 145 150155 160 Thr Val His Leu Trp Asp Val Ala Ser Gly Glu Ala Leu His Thr Phe165 170 175 Glu Gly His Thr Asp Trp Val Arg Ala Val Ala Phe Ser Pro AspGly 180 185 190 Ala Leu Leu Ala Ser Gly Ser Asp Asp Arg Thr Ile Arg LeuTrp Asp 195 200 205 Val Ala Ala Gln Glu Glu His Thr Thr Leu Glu Gly HisThr Glu Pro 210 215 220 Val His Ser Val Ala Phe His Pro Glu Gly Thr ThrLeu Ala Ser Ala 225 230 235 240 Ser Glu Asp Gly Thr Ile Arg Ile Trp ProIle 245 250 14 7 PRT Artificial Sequence katanin p80 peptide 14 Asp AlaSer Met Met Ala Met 1 5 15 5 PRT Artificial Sequence katanin p80 peptide15 Ile Gln Gly Leu Arg 1 5

What is claimed is:
 1. A kit for screening for test agents that modulatemicrotubule severing, wherein said kit comprises one or more containerscontaining an isolated microtubule severing protein consisting ofkatanin p60 subunit.
 2. The kit of claim 1, wherein said microtubulesevering protein is attached to a solid surface.
 3. The kit of claim 1,further comprising a polymerized microtubule labeled with a labelselected from the group consisting of 4′-6-diamidino-2-phenylindole(DAPI), anilinonapthalene sulfonate (ANS), bis-ANS(Bis-anilinonapthalene sulfonate), N-phenyl-1-naphthylene (NPN),ruthernium red, cresol violet, and 4-(dicyanovinyl)julolidine (DCVJ). 4.The kit of claim 3, wherein said microtubule is contacted with acompound selected from the group consisting of paclitaxel, taxotere, andguanylyl-(a,p)-methylene diphosphate (GMPCPP).
 5. The kit of claim 4,wherein said microtubule is attached to a solid surface.
 6. The kit ofclaim 5, wherein said microtubule is attached to said surface by bindingwith a motor protein.
 7. The kit of claim 3, wherein said microtubule iscontacted with paclitaxel.
 8. The kit of claim 7, wherein saidmicrotubule is attached to a solid surface.
 9. The kit of claim 8,wherein said microtubule is attached to said surface by binding with amotor protein.
 10. The kit of claim 1, further comprising a polymerizedmicrotubule labeled with 4′-6-diamidino-2-phenylindole (DAPI).
 11. Thekit of claim 10, wherein said microtubule is stabilized by contact withpaclitaxel.
 12. The kit of claim 11, wherein said microtubule isattached to a solid surface.
 13. The kit of claim 12, wherein saidmicrotubule is attached to said surface by binding with a motor protein.14. The kit of claim 10, wherein said katanin p60 subunit isrecombinant.
 15. The kit of claim 10, wherein said katanin p60 subunithas microtubule severing activity and is encoded by a nucleic acid thathybridizes under stringent conditions with a nucleic acid that encodesthe amino acid SEQ ID NO:1.
 16. The kit of claim 10, wherein saidkatanin p60 subunit has the amino acid sequence of SEQ ID NO:1.